Acidic lipids are associated with the regulation of the structure and function of membrane proteins. Therefore, accurate and highly precise analysis of acidic lipids is important for elucidating their biological roles and pathological mechanisms. In this study, an enhanced analytical method for the separation and quantification of acidic lipids, including phosphatidylserine (PS), phosphatidic acid (PA), cardiolipin, and their lyso-derivatives, was developed using nanoflow ultrahigh performance liquid chromatography-electrospray ionisation-tandem mass spectrometry. The separation and mass spectrometry detection of acidic lipids were optimised in terms of peak tailing and time-based separation efficiencies, with carbamate-embedded C18 as the stationary phase, in the presence of an appropriate liquid chromatography solvent modifier. This newly developed method was applied to analyse a lipid extract from porcine brain. A significant increase in the number of acidic lipids identified (176 vs. 134), including intact monolysocardiolipin (17 vs. 4), was observed with the new method compared with conventional C18. The quantification of acidic lipids was validated with plasma standard (NIST SRM 1950) spiked with a number of LPS and PS standards, and acceptable accuracy (<15%) was obtained. The present method was found to be reliable for the acidic lipid analysis based on qualitative results from tissue extract and plasma samples.