In numerous studies, thermosensitive liposomes (TSLs) for local heat-triggered delivery of Doxorubicin (Dox) to tumors have been investigated, with TSLs having different lipid formulations, drug loading methodology and testing procedures. To gain more insight in these parameters, we investigated TSLs with four variable DSPC-DPPC lipid ratios (50, 60, 70 or 80% DPPC and 5 mol% of DSPE-PEG2000) using either ammonium sulfate or a citrate buffer for Dox loading. Ammonium sulfate loading of Dox yielded more stable TSLs than citrate loading. At 37 °C, leakage was unnoticeable for all ammonium sulfate TSLs. At 42 °C, complete release occurred within seconds, except for 50% DPPC TSLs, where slow and incomplete release was observed in vitro but also in vivo using a dorsal skinfold window chamber. In contrast to in vitro assays, blood kinetics studies indicated a burst release of Dox upon injection and higher leakage for all TSLs. In therapeutic studies, hyperthermia in combination with TSLs repressed BFS-1 sarcoma growth. Our study shows that prediction of therapeutic efficacy purely based on differences found in vitro is difficult, instead, parameters obtained from pharmacokinetic studies in vivo, and the exact timing of the delivery protocol need to be taken into account.