Liposomal drug delivery products have been already commercialized in tumor therapeutics, which can realize passive tumor targeting via enhanced permeability and retention (EPR) effect resulting from the leaky tumor vasculature. To control drug release out of the liposomes, thermo-sensitive liposomes (TSLs) have been developed so that an abrupt exposure of highly concentrated drugs to tumor tissues was enabled by locally treated thermal stimuli. As interests upon TSL have increased along with ongoing clinical trials, some types of TSLs with different physical properties in pharmacokinetics and the mechanism of drug release have been formulated. However, there are few protocols established with a desirable heat source to maximize the efficacy of different TSLs as treating tumors. In this study, we examined different protocols for the most effective application of different TSLs to tumor therapy. First, we examined if enhancing the accumulation of TSLs within tumor tissues prior to bursting drugs out of TSLs could lead to increasing anti-tumor efficacy. Second, we compared the efficiency of two different heat sources on the use of TSL, a warm water bath (42°C) and high intensity focused ultrasound (HIFU). Our study suggests that the specified protocol be setup for TSLs with different physical properties to optimally function in tumor therapies.