Sequencing of the human genome has led to a new and even more ambitious goal – characterization of the human proteome. Such an endeavor involves not only understanding the function of hundreds of thousands of different proteins expressed in human cells, but also characterizing the millions of potential interactions that can occur with other cellular and extracellular molecules, including proteins, nucleic acids, lipids and small molecules. The ability to rapidly perform such massive glohbal proteomic screens would be a powerful tool in many areas of biotechnology including biomarker and drug discovery. Examples include the screening of a human protein library formatted on microarrays to discover new autoimmune and tumor antigens in patient sera that can be used in clinical diagnostics and immunotherapy,1–4 and the screening of a synthetic combinatorial peptide or peptoid library on beads against specific protein targets to discover new drugs.5–7 High-density protein microarrays (i.e. arrays of ‘bait’ proteins), first introduced in 2000 by MacBeath and Schreiber,8 contain as many as 10 000 distinct protein species (e.g. Life Technologies’ ProtoArray®). However, while protein microarrays can be queried with a particular ‘prey’ molecule of interest, allowing rapid identification of positive bait-prey interactions using fluorescence imaging, there are several limitations which have impeded their usefulness in the proteomics field including: (i) Low-Fidelity Readout – The inability of fluorescence alone to reveal the molecular details of the bait-prey interaction, including identification of hundreds of interacting prey species from a complex mixture and post-translational modifications to the bait proteins such as proteolysis, phosphorylation and glycosylation; (ii) Array Density, Reproducibility & Cost – Unlike DNA chips, whose probes can be synthesized at high density using photolithography, protein/peptide chips are printed using mechanical or piezoelectric devices8–10 resulting in relatively low array density, poor reproducibility of spot size, shape and uniformity, printing-induced damage to delicate proteins and relatively low throughput of the non-parallel printing process resulting in higher costs per microarray (for reviews, see9,11); (iii) Surface Chemistry & Assay Kinetics – Protein microarrays use a two-dimensional array format, with the proteins/peptides attached by passive adsorption or chemical linkage to a planar array surface and subsequently dried. While this design is ideal for readout of the chips, it results in potential surface/drying/printing induced denaturation of intact proteins,11 poor kinetics of the microarray bio-assay,12 and inefficient automation of a multitude of assays on the planar microarray ‘chips’. Screening methods that employ bait molecules immobilized on micro-particles, such as beads, are also routinely used in various proteomic and diagnostic applications.12–15 Compared to printed arrays, bead-based methods allow higher flexibility in assay design, greater number of replicates, more facile assay automation in microtiter plates and near solution-phase binding kinetics that lead to improved sensitivity and accuracy. However, these methods have limited application in proteomics since they normally employ fluorescence coding which is inherently restricted by the broad spectral emissions of fluorophores. For example, flow cytometry based multiplex methods such as the Luminex (Austin, TX, USA) xMAP® technology16 are currently limited to 500 different coded beads. In contrast to the limitations of fluorescent detection and microarrays, mass spectrometry (MS), a central tool for proteomics, has the power to detect protein modifications,17,18 perform protein identification, quantification and sequencing (e.g. using tandem mass spectrometry (MS/MS))19,20 and to detect protein–small molecule (drug) interactions.21 However, MS normally lacks the power of a microarray to localize at high density a myriad of bait molecules, and potentially interacting prey, to separate locations on a two-dimensional surface. Recently, progress in matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) has made it possible to achieve high two-dimensional spatial resolution on a sample such as a tissue slice.22 In this case, the instrument’s laser beam scans the surface of the specimen, collecting full spectra at each discrete ‘pixel’, and a multi-dimensional digital mass-image is created using the peak intensity at a given m/z value(s). In principle, the frequency tripled Nd-Yag laser beam at 355 nm used for most commercial MALDI instruments can be focused to less than 1 µm, much smaller than the diameter of most micro-beads commonly used for binding bait molecules and for combinatorial synthesis (10–100 µm).5,6 In addition, increasing scan speeds as well as improved detectors, image analysis software and automation make it feasible to apply MALDI-MSI to analyze bead libraries formatted as high-density two-dimensional bead-arrays. As demonstrated here, 30–34 µm beads incorporated at high density in a random bead-array can be spatially localized using MALDI-MSI to detect photocleavable peptide mass-tags (PC-Mass-Tags) attached to each bead. Detection with both high sensitivity and high mass resolution was achieved with a variety of bead types evaluated. Furthermore, by obtaining a fluorescence image of the same bead-array, specific fluorescently labeled beads in the array were identified and correlated with their matching PC-Mass-Tags, a process which will ultimately enable the identification of positive prey interactions with the beads.