Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry (MS) is a rapid and sensitive analytical approach that is well suited for obtaining molecular weights of peptides and proteins from complex samples. MALDI-MS can profile the peptides and proteins from single-cell and small tissue samples without the need for extensive sample preparation, except for the cell isolation and matrix application. Strategies for peptide identification and characterization of post-translational modifications are presented. Furthermore, several recent enhancements in MALDI-MS technology, including in situ peptide sequencing as well as the direct spatial mapping of peptides in cells and tissues are discussed.