Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-Tof-MS) has recently become a popular and versatile method to analyze macromolecules from biological origin. In this paper, we will review the application of MALDI-Tof-MS in clinical chemistry and biology. MALDI-Tof-MS is used in clinical chemistry, e.g. disease markers can be identified with MALDI-MS analysis in combination with 1-D and 2-D gel electrophoresis separations thanks to either peptide mass fingerprinting (PMF) or peptide sequence tag (PST) followed by data base searching. In microbiology, MALDI-Tof-MS is employed to analyze specific peptides or proteins directly desorbed from intact viruses, bacteria and spores. The capability to register biomarker ions in a broad m/z range, which are unique and representative for individual microorganisms, forms the basis of taxonomic identification of bacteria by MALDI-Tof-MS. Moreover, this technique can be applied to study either the resistance of bacteria to antibiotics or the antimicrobial compounds secreted by other bacterial species. More recently, the method was also successfully applied to DNA sequencing (genotyping) as well as screening for mutations. High-throughput genotyping of single-nucleotide polymorphisms has the potential to become a routine method for both laboratory and clinical applications. Moreover, posttranscriptional modifications of RNA can be analyzed by MALDI using nucleotide-specific RNAses combined with further fragmentation by post source decay (PSD).