Abstract: An aliquot (1.00 mL) of human urine sample was taken, 10 μL of 1.0 mg·L^-1 mixed internal standard solution (containing ¹³C-erythromycin-d₃ and amoxicillin-d₄) and 0.30 mL of Na₂EDTA-Mcllvaine buffer solution (pH 3.5±0.05) were added. The mixed solution was vortexed for 30 s and centrifuged for 5 min. The supernatant was passed through Oasis HLB solid phase extraction column at flow rate of 1.0 mL·min^-1. The column was drained, and eluted by 3 mL of methanol at flow rate of 1.0 mL·min^-1. The eluate was collected, blown to near dryness by nitrogen, and diluted to 0.5 mL by 10% (volume fraction) methanol solution. The resulting solution was vortexed and passed through a 0.22 μm organic filter membrane, after which the filtrate was analyzed by ultra-high performance liquid chromatograph-tandem mass spectrometer. Forty antibiotics (8 sulfonamides, 6 macrolides, 8 β-lactams, 4 tetracyclines, 9 quinolones, 3 chloramphenicols, 1 lincosamide and 1 quinoxaline antibiotics) were separated by gradient elution with mixed solutions composed of 0.1% formic acid (volume fraction, the same below) solution and acetonitrile solution containing 0.1% formic acid at different volume ratios on Kinetex^® F5 100A column (50 mm×3.0 mm, 2.6 μm), ionized by negative (3 chloramphenicol antibiotics) and positive ion mode of electrospray ion source, and detected by MRM mode. Erythromycin and amoxicillin were quantified by internal standard and matrix matching method, and other antibiotics were quantified by matrix matching method. As shown by the results, linear ranges of working curves of erythromycin and penicillin V were 1.0-100.0 μg·L^-1, and linear ranges of working curves of the remaining 38 antibiotics were 1.0-200.0 μg·L^-1, with detection limits (3S/N) in the range of 0.01-1.25 μg·L^-1. Test for recovery was made by standard addition method, giving recoveries in the range of 51.0%-128%, and RSDs (n=5) of the determined values ranged from 1.2% to 14%. The proposed method was applied to the analysis of 10 adult urine samples, and 9 antibiotics were detected in 9 samples, with detection amounts in the range of 0.09-322.86 μg·L^-1, of which the detection rate of amoxicillin was as high as 50% and the detection amount of that reached up to 322.86 μg·L^-1.