The detection of intact phospholipids (PLs) in sedimentary extracts using liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS) is commonly limited by ion suppression caused by complex matrices; the effect typically cannot be satisfactorily reduced using traditional column chromatographic cleanup procedures. We therefore implemented a cleanup strategy recently introduced for serum and plasma samples and adapted it for sedimentary lipid extracts. It involves selective retention of PLs on a zirconia-coated silica phase, available as a HybridSPE®-Phospholipid cartridge. Compared with conventional silica gel liquid chromatography, the HybridSPE-based protocol strongly enhances the detection of PLs using LC–ESI-MS as a result of more effective removal of complex environmental matrices, with negligible PL loss. The strength of the protocol was demonstrated by analysis of marine sediment samples from various depths and of different total organic carbon (TOC) content, for which we observed 1.2–2.7 times more intense signals from sedimentary PLs compared with the untreated reference. This new cleanup protocol significantly improves PL fingerprinting of microbial communities in sediments.