Remimazolam is a new chemical entity belonging to the benzodiazepine class of sedative drugs. A sensitive and rapid method based on ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) has been developed and validated for the determination of remimazolam and its major carboxylic acid metabolite (M1) in human urine. Urine samples were prepared by dilution and analyzed using an isocratic chromatographic separation. Inter- and intra-batch results for remimazolam were within 10.7% for accuracy and 5.5% for precision, and for M1, within 5.8% for accuracy and 4.2% for precision, respectively. This study represents the first reported example for the quantification of remimazolam and its main metabolite in human urine. Furthermore, this method has been successfully applied for the urine recovery study of remimazolam in Chinese healthy subjects. Only about 0.01% of the administered remimazolam dose was eliminated in the urine over the 24 h period in the form of unchanged remimazolam, and more than 75.1% of the administered dose was eliminated in the form of M1. Remimazolam is excreted mainly in the form of M1 in urine after intravenous administration, and there is no excessive accumulation in vivo after administration of remimazolam.