Coenzyme A (CoA) is a group of active metabolic compounds that facilitate over 100 chemical reactions in animal and plant cells. It mainly serves as an acyl carrier in many metabolic reactions and initiates the tricarboxylic acid cycle that produces more than 90% of the energy required for life processes. Measurements of short-chain and long-chain CoA compounds in a variety of tissues by using high-performance liquid chromatography (HPLC) and capillary electrophoresis-ultraviolet (CE-UV) detection have been reported, but these techniques do not allow one to simultaneously determine all the possible coexisting CoAs and their derivatives in plant tissues with sufficient sensitivity. In this paper, a method of quantitative determination of 5 short-chain CoAs in plant tissues by using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was developed. Under optimized derivatization and electrophoresis conditions, different CoAs that were derivatized with fluorescein-5-isothiocyanate (FITC) were separated and quantified at the pmole level. A fused silica capillary with a 75 µm (i.d.) × 57 cm was used for the separation and 150 mM borate buffer (pH 9.00) was used as a background electrolyte. The separation was carried out at 25 kV and completed in less than 13 min. The effects of derivatization time, buffer concentrations, and pH values on derivatization efficiency were also systematically investigated. This newly developed CE-LIF method can be used to detect CoAs in both plant and animal tissues.