Intact polar lipids (IPLs) are the building blocks of cell membranes, and amino acid containing IPLs have been observed to be involved in response to changing environmental conditions in various species of bacteria. High‐performance liquid chromatography/mass spectrometry (HPLC/MS) has become the primary method for analysis of IPLs. Many glycerol‐free amino acid containing membrane lipids (AA‐IPLs), which are structurally different than abundant aminophospholipids, have not been characterized using HPLC/MS. This results in many lipids remaining unrecognized in IPL analysis of microbial cultures and environmental samples, hampering the study of their occurrence and functionality.