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Introduction: The human pre-implantation embryo metabolism is still widely unknown. Mass spectrometry is a very sensitive proteomic method to detect the entire spectrum of molecules in samples below 1 μL. Surface Enhanced Laser Desorption Ionisation – Time Of Flight (SELDI – TOF) mass spectrometry is a further advanced method to reduce overlapping signals from different substances. Material and Methods: A total of 22 different pre-implantation embryo supernatants (10 μL each) from 16 different single-cultured embryos were analyzed. From 6 embryos, supernatants were obtained both at day 3 and 5. Embryo stage was defined by microscopy. Different stages including arrested embryos, 4- or 8-cell stage, early and expanded blastocysts were investigated. SELDI combines a chip-based separation technology with mass spectrometry. All samples were fractioned by applying six different pH milieus and then exposed to chips with six differently coated surfaces. All fractions were analyzed on a mass spectrometer type ProteinChip System, Series 4000 (Ciphergen Biosystems, Inc.). Results: By comparing the protein profiles of the analyzed embryos the implemented bioinformatic software was able to classify blinded samples correctly into groups of embryos as defined by microscopy. The use of IMAC-chips (copper-coated) and CM10-chips (cation-exchange) led to highest resolution of peaks. Although the masses of numerous molecules in culture media are assessed by this method, the exact definition of substances needs application of further techniques. Conclusions: These first SELDI mass spectrometric analyses of embryo culture media demonstrate the capability of discriminating different stages of human pre-implantation embryos by their protein expression profiles. [ABSTRACT FROM AUTHOR]