A sensitive and selective method has been developed for the determination of ritodrine diastereomers in human serum using high-performance liquid chromatography with a chiral stationary phase column and a fluorescence detector. No interfering peaks from endogenous substances were observed. The method showed good reproducibility and accuracy, and the standard curve was linear up to 100 ng/mL with a correlation coefficient of 0.999. Limit of detection (signal-to-noise = 3) and quantitation (signal-to-noise = 10) were found to be 2 and 5 ng/mL, respectively. This method is suitable for chiral pharmacological and pharmacokinetic studies as well as the therapeutic drug monitoring of ritodrine diastereomers for which no information currently exists.