Leishmaniasis is a widespread parasitic disease principally treated by intravenous drugs. Hexadecylphosphocholine (miltefosine) has recently proved its efficacy by oral route. Although its mechanism of action has been investigated, and principally relies on perturbations of the metabolism of lipids and especially phospholipids, further studies need to be conducted to detect precisely which metabolic pathways are impacted. For this purpose, the present work proposes a complete lipidomic study focused on membrane phospholipids of clones of Leishmania donovani non-treated (NT), treated (T) and resistant (R) to miltefosine. Firstly, a separation of phospholipids in normal phase high-performance liquid chromatography (NP-HPLC) was coupled to a mass spectrometer (MS) equipped with an electrospray (ESI) ion source, and response was compared to evaporative light scattering detection (ELSD). Secondly, a quantification of phospholipid classes was performed using NP-HPLC/ESI/MS on NT, T and R clones of L. donovani. Thirdly, full-scan acquisitions of analyzed samples were compared using orthogonal signal correction-partial least square-discriminant analysis (OSC-PLS-DA) to highlight phospholipid molecular species of interest between the three types of clones. Structural determination of the most relevant species has finally been performed using tandem mass spectrometry. A first hypothesis on the effect of miltefosine on lipid metabolic pathways is then proposed.