Solid-phase microextraction (SPME) is a solvent-free sample preparation technique using a fused-silica fiber coated with an appropriate stationary phase. Derivatization is often necessary to determine substances with poor chromatographic behavior, high reactivity, and/or volatility or thermal instability. SPME derivatization technique can be operated under different combined modes and may be influenced by factors such as sample matrix, SPME, derivatization, and desorption condition. Combined with derivatization, HS-SPME, DI-SPME, membrane-protected SPME, and in-tube SPME have been applied to the analysis of a vast amount of chemicals, i.e., aldehydes and acetone, amines, organic acids, phenolic compounds, pesticide, metal and organic metal species, and pharmaceutical and personal care products (PPCPs) in a wide range of environmental matrices, including air, water, soil, and sediment samples. This chapter reviews the mode and method development of the derivatization technique, the derivatization reagents, and reaction mechanism, as well as its applications in environmental analysis.