In this work, phospholipids extracted from egg yolk (control group, experimental group) were identified using high performance liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS). Combinations of fatty acyls occurring in 11 classes of phospholipids from egg yolk were investigated. Differences between the profile of fatty acyls from hens fed traditionally and the ones that received special diet supplementation were observed. Experimental findings were complemented with multivariate chemometric analysis. Multiple reaction monitoring mass spectrometry mode was utilized and 123 distinct combinations of fatty acyls occurring in phospholipids were identified. From these, large portions are polyunsaturated fatty acyls from the omega-3 and omega-6 family. HPLC MS/MS analysis allows for quick, accurate and precise determination of biologically active compounds, found in low concentrations within the tested material.