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Purpose: Early diagnosis of primary immunodeficiency disorders (PIDDs) is critical for maximizing patient survival and clinical outcomes. Consequently, there is significant interest in developing broad-based, high-throughput, screening approaches capable of utilizing small blood volumes to identify patients with PIDD., Experimental Design: We developed a novel proteomic screening approach using tandem mass spectrometry to simultaneously identify specific signature peptides derived from the transmembrane protein cluster of differentiation 3 (CD3)ɛ and the intracellular proteins Wiskott-Aldrich syndrome protein (WASP) and Bruton's tyrosine kinase (BTK) as markers of three life-threatening PIDDs; severe combined immunodeficiency, Wiskott-Aldrich syndrome, and X-linked Agammaglobulinemia. Signature peptides were analyzed by LC/MS-MS in proteolytically digested lysates from cell lines and white blood cells (WBCs). The amount of each peptide was determined by the ratio of the signature peptide peak area to that of a known amount of labeled standard peptide. Peptide concentrations were normalized to actin., Results: We show that signature peptides from CD3ɛ, WASP, and BTK were readily detected in proteolytically digested cell lysate and their absence could correctly identify PIDD patients., Conclusions and Clinical Relevance: This proof of concept study demonstrates the applicability of this approach to screen for PIDD and raises the possibility that it could be further multiplexed to identify additional PIDDs and potentially other disorders., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)