In recent years, there has been an increasing interest in nitro fatty acids (NO2-FA) as signaling molecules formed under nitroxidative stress. NO2-FA were detected in vivo in a free form, although it is assumed that they may also be esterified to phospholipids (PL). Nevertheless, insufficient discussion about the nature, origin, or role of nitro phospholipids (NO2-PL) was reported up to now. The aim of this study was to develop a mass spectrometry (MS) based approach which allows identifying nitroalkenes derivatives of three major PL classes found in living systems: phosphatidylcholines (PCs), phosphatidylethanolamine (PEs), and phosphatidylserines (PSs). NO2-PLs were generated by NO2BF4 in hydrophobic environment, mimicking biological systems. The NO2-PLs were then detected by electrospray ionization (ESI-MS) and ESI-MS coupled to hydrophilic interaction liquid chromatography (HILIC). Identified NO2-PLs were further analyzed by tandem MS in positive (as [M + H]+ ions for all PL classes) and negative-ion mode (as [M – H]− ions for PEs and PSs and [M + OAc]− ions for PCs). Typical MS/MS fragmentation pattern of all NO2-PL included a neutral loss of HNO2, product ions arising from the combined loss of polar headgroup and HNO2, [NO2-FA + H]+ and [NO2-FA – H]− product ions, and cleavages on the fatty acid backbone near the nitro group, allowing its localization within the FA akyl chain. Developed MS method was used to identify NO2-PL in cardiac mitochondria from a well-characterized animal model of type 1 diabetes mellitus. We identified nine NO2-PCs and one NO2-PE species. The physiological relevance of these findings is still unknown.