Lipidomics has emerged as a powerful technique to study cellular lipid metabolism. As the lipidome contains numerous isomeric and isobaric species resulting in a significant overlap between different lipid classes, cutting edge analytical technology is necessary for a comprehensive analysis of lipid metabolism. Just recently differential mobility spectrometry (DMS) has evolved as such a technology, helping to overcome several analytical challenges. We here set out to apply DMS and the LipidyzerTM platform to obtain a comprehensive overview of leukocyte related lipid metabolism at resting and activated state. First we tested linearity and repeatability of the platform by using HL60 cells. We obtained good linearities for most of the thirteen analyzed lipid classes (correlation coefficient > 0.95), and good repeatability (%CV < 15). By comparing the lipidome of neutrophils (PMNs), monocytes (CD14+), and lymphocytes (CD4+) we shed light on leukocyte specific lipid patterns as well as lipidomic changes occurring through differential stimulation. For example, at resting state PMNs proved to contain higher amounts of triacylglycerides compared to CD4+ and CD14+ cells. On the other hand, CD4+ and CD14+ cells contained higher levels of phospholipids and ceramides. Upon stimulation, diacylglycerides, hexosylceramides, phosphatidylcholines, phosphoethanolamines, and lysophosphoethanolamines were upregulated in CD4+ and PMNs, whereas CD14+ cells did not show significant changes. By exploring fatty acid content on the significantly upregulated lipid classes we found mainly increased concentrations of very long and poly-unsaturated fatty acids. Our results show the usefulness of the LipidyzerTM platform for studying cellular lipid metabolism. Its application allowed us to shed light on the lipidome of leukocytes.