Matrix-assisted laser desorption/ionisation (MALDI) time-of-flight (TOF) mass spectrometry is an important analytical tool in chemistry and biology. Bearing the advantages of easy-to-use, fast measurements and relatively simple data interpretation, MALDI-TOF mass spectrometry has been expanded from research laboratories to clinical applications. In this thesis, methods based on MALDI-TOF mass spectrometry are developed for the purpose of bacterial infection diagnosis and cancer analysis. Magnetic beads coated with antibodies specific to bacterial species are used to isolate and enrich bacterial cells from complex matrices. This immuno-affinity bacterial capture/enrichment procedure is combined with MALDI-TOF mass spectrometry for the fast identification of bacterial pathogens in human blood. Titanium dioxide nanoparticles-modified target plates are designed to improve the MALDI efficiency for measuring bacterial cellular components by extending the mass range and lowering the detection sensitivity. This titanium dioxide-facilitated MALDI-TOF mass spectrometry method allows the detection of antimicrobial resistance-associated proteins directly from intact bacterial cells. A combination of immuno-affinity enrichment strategy and redox indicators-mediated amperometric read-out is implemented in a point-of-care platform for bacteria detection. The platform provides bacterial species determination, living cell quantification and antimicrobial susceptibility testing. It could serve as a supplement to MALDI-TOF mass spectrometry to speed up bacterial infection diagnosis. MALDI-TOF mass spectrometry detection of exosomes is proposed as a potential tool for cancer analysis. It allows the classification of cancer cells, tracking of target proteins, detection of cancer biomarkers and exploring cancer progression via exosomes-mediated intercellular communication. Targeting blood-circulating exosomes, it also opens new ways for fast detection of cancer diseases and dynamic monitoring of the disease status. Cells are recovered from human skin using adhesive tapes, and the skin cells-bearing tapes are directly transferred onto a MALDI target for skin cell fingerprinting using MALDI-TOF mass spectrometry. This tape stripping mass spectrometry approach is proposed for rapid and non-invasive detection of skin diseases like skin cancers.