Ionic liquids (ILs) are entirely composed of ions and they possess fascinating properties, including low volatility, tunable viscosity, miscibility and electrolytic conductivity, which make them promising alternatives to traditional organic solvents used in sample preparation. The recent surge in the number of publications clearly indicates an increasing interest of the analytical and bioanalytical community toward these exciting and unique solvents. This article highlights the recent advances in the use of ILs as extraction solvents, as materials for separation and preconcentration in chromatographic techniques, and as matrices in mass spectrometric techniques for bioassays in biocomplex samples. We also briefly discuss the potential applications of ILs in biocatalysis.