Proper　chromosome　segregation　during　cell　division　relies　on　the　timely　dissolution　of　chromosome　cohesion.　Separase　(EC　3.4.22.49),　a　cysteine　protease,　plays　a　critical　role　in　mitosis　by　cleaving　the　kleisin　subunit　of　cohesin,　thereby　presenting　a　promising　target　for　cancer　therapy.　However,　challenges　in　isolating　active　human　separase　suitable　for　high-throughput　screening　have　limited　the　identification　of　effective　inhibitors.　Here,　we　conducted　a　high-throughput　screening　of　small-molecule　inhibitors　using　the　protease　domain　of　Chaetomium　thermophilum　separase　(ctSPD),　which　not　only　shares　significant　sequence　similarity　with　human　separase　but　is　also　readily　available.　After　conducting　a　primary　screening　of　a　library　containing　9,172　compounds　and　subsequent　validation　using　human　separase,　we　identified　walrycin　B　and　its　analogs,　toxoflavin,　3-methyltoxoflavin,　and　3-phenyltoxoflavin,　as　potent　inhibitors　of　human　separase.　Subsequent　microscale　thermophoresis　assays　and　molecular　dynamics　simulations　revealed　that　walrycin　B　binds　to　the　active　site　of　separase　and　competes　with　substrates　for　binding.　Additionally,　cell-based　studies　showed　that　walrycin　B　and　its　analogs　effectively　induce　cell　cycle　arrest　at　the　M　phase,　activate　apoptosis,　and　ultimately　lead　to　cell　death　in　mitosis.　Finally,　in　a　mouse　xenograft　model,　walrycin　B　exhibited　significant　antitumor　efficacy　with　minimal　side　effects.　Together,　these　findings　highlight　the　therapeutic　potential　of　walrycin　B　for　cancer　treatment　and　its　utility　as　a　chemical　tool　in　future　studies　involving　separase.　©　2024　Elsevier　Inc.