Fibroblast activation protein-α (FAPα) is a tumor-associated antigen uniquely expressed by reactive stromal fibroblasts in the majority of human epithelial tumors. FAPα also possesses both post-prolyl peptidase and endopeptidase activities. Consequently, FAPα is increasingly considered as a potential pan-tumor target for designing tumor-targeted prodrugs. We previously conjugated Doxorubicin (Dox) with a FAPα-specific dipeptide (Z-Gly-Pro) to develop a FAPα-targeting prodrug of Dox (FTPD). The aim of current work was to validate the tumor targeting of this targeted-delivery strategy. The results demonstrated that FTPD could effectually release Dox upon the hydrolysis of FAPα as well as the incubation with tumor homogenate of FAPα-positive tumor (4T1 tumor), while it was highly stable in mouse plasma and a variety of tissue homogenates including heart, liver, and so on. And the FAPα-cleaved FTPD exhibited significantly higher cytotoxicity against 4T1 cells in vitro than the uncatalyzed prodrug. Additionally, FTPD produced similar antitumor efficacy in 4T1 tumor-bearing mice to free Dox without obvious cardiotoxic effect. Moreover, subsequent study indicated that the accumulation of FTPD reduced significantly in the heart compared to free Dox. These findings suggest that such FAPα-based prodrug strategy is promising to achieve targeted delivery of antitumor agents.