Lipids play fundamental roles in mammalian embryo preimplantation development and cell fate. Triacylglycerol accumulates in oocytes and blastomeres as lipid droplets, phospholipids influence membrane functional properties, and essential fatty acid metabolism is important for maintaining the stemness of cells cultured in vitro. The growing impact that lipids have in the field of developmental biology makes analytical approaches to analyse structural information of great interest. This paper describes the concept and presents the results of lipid profiling by mass spectrometry (MS) of oocytes and preimplantation embryos, with special focus on ambient ionisation. Based on our previous experience with oocytes and embryos, we aim to convey that ambient MS is also valuable for stem cell differentiation analysis. Ambient ionisation MS allows the detection of a wide range of lipid classes (e.g. free fatty acids, cholesterol esters, phospholipids) in single oocytes, embryos and cell pellets, which are informative of in vitro culture impact, developmental and differentiation stages. Background on MS principles, the importance of underused MS scan modes for structural analysis of lipids, and statistical approaches used for data analysis are covered. We envisage that MS alone or in combination with other techniques will have a profound impact on the understanding of lipid metabolism, particularly in early embryo development and cell differentiation research.