Abstract: A new assay has been developed to analyze the secretome of human SH-SY5Y by OFFGEL and LC-MS combined with the software of bioinformatics. The proteins collected from the extracellular SH-SY5Y mediums have been digested and fractionated by the OFFGEL, and then different fractions were performed by reverse phase liquid chromatography with electrospray tandem mass spectrometry. The 88 potential secretory proteins from Mascot results were predicted and screened by the bioinformatics software called SignalP. Only 31 proteins have been confirmed by the SingalP. Based on the neural networks and hidden Markov models of SignalP, which could increase the identification probability, it was found that the CERU_HUMAN was one of secretory proteins with the location of signal peptide cleavage sites between 19 and 20 amino acid. The new assay would play more and more important role in the application of secretome analysis.