Liposomes are potent nanocarriers to deliver chemotherapeutic drugs to tumors. However, the inefficient drug release hinders their application. Thermosensitive liposomes (TSL) can release drugs upon heat. This study aims to identify the optimum 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG(2000) (DSPE-PEG(2000)) concentration in stealth TSL to improve content release efficiency under mild hyperthermia (HT). TSL were prepared with DSPE-PEG(2000) from 1 to 10 mol%, around 80 nm in size. Quenched carboxyfluorescein (CF) in aqueous phase represented encapsulated drugs. In vitro temperature/time-dependent CF release and TSL stability in serum were quantified by fluorometry. In vivo CF release in dorsal skin flap window chamber models implanted with human BLM melanoma was captured by confocal microscopy. In vitro heat triggered CF release increased with increasing DSPE-PEG(2000) density. However, 6 mol% and higher DSPE-PEG(2000) caused CF leakage at physiological temperature. TSL with 5 mol% DSPE-PEG(2000) were stable at 37 degrees C, while released 60% CF in 1 min and almost 100% CF in 1h at 42 degrees C. In vivo optical intravital imaging showed immediate massive CF release above 41 degrees C. In conclusion, incorporation of 5 mol% DSPE-PEG(2000) optimized stealth TSL content release triggered by HT.