Secretory phospholipase A2 (sPLA2) expression is increased in several cancers and has been shown to trigger release from some lipid carriers. This study used electrospray ionization mass spectrometry (ESI‐MS) and release of 6‐carboxyfluorescein (6‐CF) to determine the effects of sPLA2 on various liposome formulations. Different combinations of zwitterionic [1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphatidylcholine, 1,2‐distearoyl‐sn‐glycero‐3‐phosphatidylcholine, and 1,2‐ distearoyl‐sn‐glycero‐3‐phosphatidylethanolamine (DSPE)] and anionic [1,2‐distearoyl‐sn‐glycero‐3‐phosphatidic acid, 1,2‐distearoyl‐sn‐glycero‐3‐phosphatidylglycerol (DSPG), 1,2‐distearoyl‐sn‐glycero‐3‐phosphatidylserine, and 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine–N‐poly(ethylene glycol) 2000 (DSPE–PEG)] phospholipids were examined. DSPG and DSPE were most susceptible to sPLA2‐mediated degradation compared with other phospholipids. Increased 6‐CF release was observed after inclusion of 10 mol % DSPE and anionic lipids into different liposome formulations. Group IIa sPLA2‐mediated 6‐CF release was less than Group III and relatively insensitive to cholesterol (Chol), whereas Chol reduced sPLA2‐mediated release. Inclusion of DSPE–PEG increased sPLA2‐mediated 6‐CF release, whereas serum reduced lipid degradation and 6‐CF release significantly. These data demonstrate that ESI‐MS and 6‐CF release were useful in determining the selectivity of sPLA2 and release from liposomes, that differences in the activity of different sPLA2 isoforms exist, and that DSPE–PEG enhanced sPLA2‐mediated release of liposomal constituents. These findings will aid in the selection of lipids and optimization of the kinetics of drug release for the treatment of cancers and diseases of inflammation in which sPLA2 expression is increased. © 2011 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3146–3159, 2011