Ionic liquids (ILs) have advanced a variety of applications, including matrix-assisted laser desorption/ionization–mass spectrometry (MALDI–MS). ILs can be used as matrices and solvents for analyte extraction and separation prior to analysis using laser desorption/ionization–mass spectrometry (LDI–MS). Most ILs show high stability with negligible sublimation under vacuum, provide high ionization efficiency, can be used for qualitative and quantitative analyses with and without internal standards, show high reproducibility, form homogenous spots during sampling, and offer high solvation efficiency for a wide range of analytes. Ionic liquids can be used as solvents and pseudo-stationary phases for extraction and separation of a wide range of analytes, including proteins, peptides, lipids, carbohydrates, pathogenic bacteria, and small molecules. This review article summarizes the recent advances of ILs applications using MALDI–MS. The applications of ILs as matrices, solvents, and pseudo-stationary phases, are also reviewed.