Recently, colistin has become one of the most important drugs for treating infections caused by multidrug-resistant Gram-negative bacteria. Therapeutic drug monitoring is recommended to ensure the safety and efficacy of colistin and to improve clinical outcomes. This study developed an accurate and sensitive high-performance liquid chromatography-fluorescence detection (HPLC-FLD) method for the quantification of colistin in human plasma. The sample preparation included protein precipitation using trichloroacetic acid (TCA) and methanol, followed by in-solid phase extraction (In-SPE) derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl). A Poroshell 120 EC-C18 2.1×100mm (2.7μm) column was used in the HPLC method with a mobile phase composed of acetonitrile (ACN), tetrahydrofuran (THF), and deionized (DI) water (82%, 2%, 16% (v/v), respectively). Polymyxin B1 was used as the internal standard. The total analysis time was 22min under optimal separation conditions. The HPLC-FLD method was validated over a therapeutic range of 0.3-6.0μgmL(-1). The intra-day and inter-day precisions for colistin A and colistin B were below 9.9% and 4.5% relative standard deviations, respectively. The accuracy test results were between 100.2 and 118.4%. The extraction recoveries were between 81.6 and 94.1%. The method was linear over the test range, with a 0.9991 coefficient of determination. The limit of detection was 0.1μgmL(-1). The validated HPLC-FLD method was successfully applied to quantify the colistin concentrations in 2 patient samples for therapeutic drug monitoring.