Human TROP2 protein was not detected, but strong luminescence and GFP fluorescence were observed in B-Luc-GFP KI, TROP2 KO BxPC-3 cells. TROP2-ADC treatment led to targeted killing of TROP2-positive and bystander killing of TROP2-negative B-Luc-GFP KI, TROP2 KO cells, both in vitro and in vivo. The B-Luc-GFP KI, TROP2 KO BxPC-3 cell model is a reliable tool for preclinical evaluation of the bystander effect of ADC therapeutics.