OBJECTIVE： To establish a method for the simultaneous determination of procaine， benzathine and penicillin in Triple penicillin for injection. METHODS： HPLC was performed on the column of SinoChrom C18 with mobile phase of 0.21 mol/L potassium dihydrogen phosphate solution（containing 0.16％ triethylamine， adjusted pH to 5.6 by potassium hydroxide）-acetonitril （81 ∶ 19，V/V） at a flow rate of 1.0 mL/min， detection wavelength was 215 nm， column temperature was 25 ℃， and the injection volume was 10 μL. RESULTS： The linear range was 0.011-0.64 mg/mL for procaine （r＝0.999 9）， 0.013-0.80 mg/mL for benzathine（r＝0.999 9） and 0.075-4.50 mg/mL for penicillin （r＝0.999 9）； RSDs of precision， stability and reproducibility tests were lower than 0.70％； recoveries were 98.42％-101.51％ （RSD＝0.90％，n＝9）， 98.28％-101.50％ （RSD＝0.90％，n＝9） and 99.03％-101.11％ （RSD＝0.66％，n＝9）. CONCLUSIONS： The method is simple， accurate， and can be used for the simultaneous determination of procaine， benzathine and penicillin in Triple penicillin for injection.