Nanoassemblies (NAs) of small-molecule lipophilic prodrugs have been widely investigated for efficient drug delivery in cancer therapy, but their kinetic stability has not attracted sufficient attention in the past studies. Herein, we reported that kinetic stability has a great influence on the drug release from the NAs of lipophilic prodrugs in physiologically relevant media. Based on the co-assembled FRET nanosystems of two lipophilic fluorescent prodrugs, we demonstrated that NAs constructed by lipophilic prodrugs containing shorter alkyl chains or those with higher unsaturated degrees displayed poorer kinetic stability, which further resulted in remarkably faster drug release in mouse plasma and various tissue homogenates. More importantly, these kinetically unstable NAs also induced rapid intracellular drug release, resulting in much more potent cytotoxicity. These findings highlight the crucial role of kinetic stability in determining the drug release from the NAs of lipophilic prodrugs, which would effectively guide their rational designs for cancer therapy.