Sample preparation is essential for isolating desired components from complex matrices and greatly influences their reliable and accurate analysis. Solid-phase microextraction (SPME) is a new and effective sample preparation technique. Fibers and capillary tubes coated with an appropriate stationary phase are usually used for SPME, but alternative microextraction techniques, including solid-phase dynamic extraction using an internal coated needle, microextraction in a packed syringe and stir-bar-sorptive extraction using a coated magnetic stir bar, have been developed recently. These techniques, in combination with gas chromatography (GC), GC-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), LC-MS or capillary electrophoresis, can be used for analysis for complex mixtures. These microextraction techniques save preparation time, as well as solvent purchase and disposal costs. This review summarizes recent advances in SPME and related microextraction techniques and their applications in pharmaceutical and biomedical analysis.