Abstract: A method was proposed for the simultaneous determination of 16 sulfonamides (such as N-sulfanilylacetamide, sulfadiazine and sulfathiazole) as well as 6 macrolides (such as spiramycin, azithromycin and erythromycin) in animal urine by QuEChERS purification combined with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). An aliquot (2 mL) of urine sample was taken, and 0.1 mL of formic acid was added. After vortex for 10 s, 4 mL of acetonitrile and 2 g of sodium chloride were added, and the mixture was shaken for 5 min, and centrifuged for 5 min. The 2 mL of supernatant was taken and added into to a 15 mL-centrifuge tube containing 50 mg of octadecyl bonded silica gel (C₁₈), 50 mg of ethylenediamine-N-propylsilane (PSA), and 50 mg of functionalized polystyrene/divinylbenzene (PEP). The mixture was vortexed for 10 s and centrifuged for 5 min, and 1 mL of the supernatant was taken and blown to dryness by nitrogen at 40 ℃. The mixed solution (1 mL) composed of acetonitrile and water at volume ratio of 1∶9 was added, and the mixture was vortexed for 10 s, and passed through a 0.22 μm organic filter membrane. The filtrate was determined according to the operating conditions of the instrument. The 22 antibiotics were separated on Kinetex XB-C₁₈ column by gradient elution using the mixed solution of methanol and 0.1% (volume fraction) formic acid solution containing 5 mmol·L⁻¹ ammonium acetate at different volume ratios as mobile phase, ionized by positive ion mode of electrospray ion source, and detected by multiple reaction monitoring (MRM) mode, with matrix matching method for quantification. It was shown that linear relationships between values of the mass concentration and the peak area of 22 antibiotics were kept in definite ranges, with detection limits (3S/N) in the range of 0.2-1.5 μg·L⁻¹. At spiked concentrations of 5, 10, 50 μg·L⁻¹, recoveries ranged from 67.1% to 114%, giving RSDs (n=6) of the determined values in the range of 4.1%-12%. The proposed method was used for the analysis of animal urine samples from livestock farms, and the detected antibiotics were mainly N-sulfanilylacetamide (with detection amounts in the range of 22.7-28.5 μg·L⁻¹) and sulfachloropyridazine (with detection amounts in the range of 11.3-75.6 μg·L⁻¹).