The protein tyrosine phosphatase Src homology phosphotyrosyl phosphatase 2(SHP2)is implicated in various cancers,and targeting SHP2 has become a promising therapeutic approach.We herein described a robust cross-validation high-throughput screening protocol that combined the fluorescence-based enzyme assay and the conformation-dependent thermal shift assay for the discovery of SHP2 inhibitors.The established method can effectively exclude the false positive SHP2 inhibitors with fluorescence interference and was also successfully employed to identify new protein tyrosine phos-phatase domain of SHP2(SHP2-PTP)and allosteric inhibitors.Of note,this protocol showed potential for identifying SHP2 inhibitors against cancer-associated SHP2 mutation SHP2-E76A.After initial screening of our in-house compound library(～2300 compounds),we identified 4 new SHP2-PTP inhibitors(0.17％hit rate)and 28 novel allosteric SHP2 inhibitors(1.22％hit rate),of which SYK-85 and WS-635 effectively inhibited SHP2-PTP(SYK-85:IC50=0.32 μmol/L;WS-635:IC50=4.13 μmol/L)and thus represent novel scaffolds for designing new SHP2-PTP inhibitors.TK-147,an allosteric inhibitor,inhib-ited SHP2 potently(IC50=0.25 μmol/L).In structure,TK-147 could be regarded as a bioisostere of the well characterized SHP2 inhibitor SHP-099,highlighting the essential structural elements for allosteric inhibition of SHP2.The principle underlying the cross-validation protocol is potentially feasible to iden-tify allosteric inhibitors or those inactivating mutants of other proteins.