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Aim: A high-throughput screening model was established on 384-well microplate with 50 μL volume in order to screen potential human soluble protein tyrosine phosphatase 1B (PTP1B) inhibitors.Methods: Recombinant PTP1B was cloned and expressed in E.coli.the specific phosphotyrosine peptides substrate [TRDIY(P) ETDYYRK, PPS], base on the insulin receptor (GenBank: ADQ39592.1) 371-381 amino acid sequence, was syntheticed; A chromogenic assay based on the Malachite Green method was employed for the detection of inorganic phosphate (Pi) released from phospho-peptides by PTP1B.