Matrix-assisted laser desorption and ionization (MALDI) mass spectrometry (MS) - often but not exclusively combined with time-of-flight (TOF) mass analyzers - is one of the most successful “soft” ionization methods that is applicable to a very broad range of analytes. Although the ionization process is still widely unknown and a matter of intense research, it is commonly accepted that the matrix plays a crucial role. The development of and the search for useful matrix compounds, however, was - and still is - an empirical process because only some obvious properties (vacuum stability, high absorption at the laser wavelength, etc.) that must be fulfilled by a compound to become a useful MALDI matrix are known so far. Although MALDI MS is primarily used for the analysis of polar biopolymers, such as proteins and nucleic acids, the focus of this review is on the analysis of apolar molecules with special emphasis on lipids and phospholipids. Beside established organic matrix molecules, such as cinnamic or benzoic acid derivatives, the advantages and drawbacks of liquid crystalline matrices and mixtures of common matrices will be also shortly discussed. It will be shown that only the careful selection of the matrix enables the detection of the compounds of interest. It will also be demonstrated that mixture analysis and the quantitative analysis of small molecules can be accurately performed only if the matrix is carefully selected. Finally, some basic principles how useful matrix compounds can be de novo “designed” will be introduced.