Gilteritinib, an oral inhibitor of FMS-like tyrosine kinase 3 (FLT3), is a standard treatment for FLT3-mutated acute myeloid leukemia. We developed a simple high-performance liquid chromatography-ultraviolet (HPLC-UV)-based method for determining the concentration of gilteritinib in human plasma. The analysis requires extracting a 200-μL plasma sample and the precipitation of proteins by solid-phase extraction. Gilteritinib was isocratically separated within 10 min using a mobile phase of acetonitrile:0.5% monopotassium phosphate (KH2 PO4 , pH 3.5; 28:72, v/v) on a Capcell Pack C18 MG II (250 mm × 4.6 mm) column at a flow rate of 1.0 mL/min and monitored at 250 nm. The calibration curve was found to be linear within a plasma concentration range of 25-2500 ng/mL, with the coefficient of determination (r2 ) being 0.9997. The coefficients of intra-day and inter-day validation were 2.3-3.7% and 1.3-5.2%, respectively. The accuracy of the assay and recovery were -9.6 to 0.1% and > 81.8%, respectively. This HPLC-UV method for determining the plasma concentration of gilteritinib is simple and can be effectively applied to routine drug monitoring.