We recently published a new concept using monoacylglycerol-like fragments [MG+H-H2O]+ (ions B) pro-duced in-source by atmospheric pressure photoionization in positive mode and high-resolution massspectrometry for the determination of the fatty acyl (FA) composition of triacylglycerols (TGs) from plantoils. This study extends the concept to the phospholipids (PLs) category and shows that the APCI+ sourcecan also be used. Moreover, the coupling with NP-LC allows to simultaneously analyze different PLsclasses in the same sample. We compared the relative intensities of the ions B produced in-source tothe % composition of FAs determined by GC-FID. In the case of PLs from natural extracts composed ex-clusively of diacyl-PLs, the relative intensities of ions B are close to the % of the FAs obtained by GC-FID.This approach is not directly useable for extracts containing plasmalogens (P-PLs). For these PLs, acidichydrolysis by HCl fumes allows hydrolyzing selectively vinyl ether functions to form lyso-PLs. The analy-sis of hydrolyzed extracts makes it possible to obtain the composition of P-PLs FAs thanks to the lyso-PLsthus formed, while the diacyl-PLs composition remains unchanged. Unlike GC-FID FAs determination, thisapproach allows a distinction between the diacyl-PLs and P-PLs FAs composition. We also found that theion B intensities were consistent among the PL classes (PG, PE, PA, PI, CL, PS and PC) and lyso- forms(LPE and LPC). In the case of the diacyl-PLs extracts analyzed, no statistically significant differences werefound between the PLs FAs compositions calculated from ion B intensities and the corresponding GC-FIDdata. A weighting coefficient was applied to correct ion B intensities issued from polyunsaturated FAswith three or more double bonds. The fatty alkenyls composition of P-PLs could also be calculated fromthe % intensities of specific ions.