Abstract The overexpression of secretory phospholipase A₂ (sPLA₂) in tumors has opened new avenues for enzyme-triggered active unloading of liposomal antitumor drug carriers selectively at the target tumor. However, the effects of the liposome composition, drug encapsulation, and tumor microenvironment on the activity of sPLA₂ are still not well understood. We carried out a physico-chemical study to characterize the sPLA₂-assisted breakdown of liposomes using dye-release assays in the context of drug delivery and under physiologically relevant conditions. The influence of temperature, lipid concentration, enzyme concentration, and drug loading on the hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, T_m = 42°C) liposomes with snake venom sPLA₂ was investigated. The sensitivity of human sPLA₂ to the liposome composition was checked using binary lipid mixtures of phosphatidylcholine (PC) and phosphatidylglycerol (PG) phospholipids with C14 and C16 acyl chains. Increasing temperature (36-41°C) was found to mainly shorten the enzyme lag-time, whereas the effect on lipid hydrolysis rate was modest. The enzyme lag-time was also found to be inversely dependent on the lipid-to-enzyme ratio. Drug encapsulation can alter the hydrolysis profile of the carrier liposomes. The activity of human sPLA₂ was highly sensitive to the phospholipid acyl-chain length and negative surface charge density of the liposomes. We believe our work will prove useful for the optimization of sPLA₂-susceptible liposomal formulations as well as will provide a solid ground for predicting the hydrolysis profile of the liposomes in vivo at the target site.