Matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF-MS) is widely used in a variety of fields because it has the characteristics of speed, ease of use, high sensitivity, and wide detectable mass range for obtaining molecular weights and for structural characterization of macromolecules. In this article we summarize recent developments in matrix additives, new matrices, and sample-pretreatment methods using off-probe or on-probe techniques or nanomaterials for MALDI-TOF-MS analysis of biological samples. [ABSTRACT FROM AUTHOR]