The invention relates to a method for determining the content of antibiotics, and in particular to a method for determining the content of 52 kinds of antibiotic drugs in children. The method is characterized by comprising the following steps of: preparation of a mixed standard control solution, pretreatment of a sample to be detected, pretreatment of a spiked sample, pretreatment of a blank sample, qualitative analysis and quantitative analysis. The method disclosed by the invention has the beneficial effects that the accurate content of the antibiotic drugs in children are determined throughthe qualitative and quantitative analysis on the 52 kinds of antibiotic drugs; specific ion pairs generated in the substance to be detected can be detected with high selectivity and without the interference of other substances in plasma or urine; and the detection method is simple in steps, accurate in measurement, high in sensitivity, low in detection limit and short in measurement time, the lower limit of quantitation can be up to 0.5 ng/mL and the time from sample receiving to data obtaining is only 30 minutes.