This study was designed to develop hypothetical inhibition mechanism of novel UTB inhibitor and exploit novel compounds with UTB inhibitory activity and to obtain promising lead compounds.We integrated cell based high throughput screening and in silico method to identify an undiscovered UTB inhibitor binding site and proposed the mechanism of UTB inhibitor in crossspecies.We employed highthroughput screening using an erythrocyte osmotic lysis assay and identified 4 compounds PU21, PU168, PU468 and PU474 with UTB inhibitory activity in vitro from 2319 hits.16 compounds with UTB inhibitory activity were screened by erythrocyte osmotic lysis assay from 60 analogues of PU21.PU14, one of 16 compounds exhibited potential inhibition activity in human, rabbit,rat, mouse in vitro and pharmacological diuresis activity in vivo.Based on the physiological data, we built a computational mode of human UTB by homology modeling.The putative UTB binding site was identified by structurebased drug design and validated by ligandbased and QSAR model.Additionally, UTB structural and functional differences under inhibitors treated and untreated conditions were simulated by Molecular Dynamics (MD).The UTB inhibitor binding site analysis and validation provide structure basses for lead identification and optimization.