The important roles of lipids particularly certain phospholipids in signal transduction processes and as important disease markers are becoming increasingly evident. Unfortunately, however, sensitive methods of lipid analysis are established to a much lesser extent than, e.g., methods of protein analysis. Mass spectrometry (MS) is an increasingly used technique of lipid analysis and electrospray ionization (ESI) MS is the so far most established ionization method. Although matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) was so far primarily used for protein analysis, however, this method has itself proven to be very useful in the field of lipid analysis, too. This chapter gives an overview of methodological aspects of MALDI-TOF MS in lipid research and summarizes the specific advantages and drawbacks of this soft-ionization method. In particular, suppression effects of some lipid classes, especially those with quaternary ammonia groups such as phosphatidylcholine, will be highlighted and possible ways to overcome this problem (use of different matrices, separation of the relevant lipid mixture prior to analysis) will be discussed on the example of an organic liver extract.