OBJECTIVE To develop a new clysma preparation of diazepam rectal delivery system for pediatrics and to determine the content of diazepam in the preparation.METHODS By selection of the formula and the process, diazepam was dissolved in the co-solvent system. The stability of diazepam rectal clysma was investigated under the conditions of room temperature, high temperature(40℃,60℃), strong light(4 500 lux) etc..The content of diazepam in the rectal clysma was determined by a reversed phase high performance liquid chromatography.The mobile phase was consisted of methanol and water (70∶30).The ultraviolet detection wavelength was at 254 nm and the naphthalene was used as the internal standard.RESULTS The propylene glycol co-solvent played a key role for stabilization.The standard curves were obtained with a liner range of 25～350 mg·L^-1 (r=0.999 98,n=5).The recovery was from 98.97% to 100.45%, the average relative standard deviation of intra- and inter-day were 1.76% and 0.65%,respectively.CONCLUSION The preparation of diazepam rectal clysma was stable,convenient and feasible. The established HPLC-UV method can be used for daily determination of diazepam rectal clysma and other preparations of the diazepam.