A high performance liquid chromatography procedure has been developed for the assay of an ondansetron hydrochloride and diphenhydramine hydrochloride mixture in 0.9% sodium chloride injection. The separation and quantitation were achieved on a 5-μm Spherisorb ODS-1 column at ambient temperature using a mobile phase of 60:40 v/v 0.1 M phosphate buffer pH 4.5-acetonitrile at flow rate of 1.2 mL/min. with detection of both analytes at 210 nm. The separation was achieved within 22 min. with sensitivity in the ng/mL range for each analyte. The method showed linearity for ondansetron and diphenhydramine in the 0.40–6.40 and 5.0–80.0 μg/mL ranges, respectively. Intra- and inter-day RSD values were 1.8% and 2.8–3.8% for ondansetron, and 1.4–1.7% and 2.0–2.7% for diphenhydramine, respectively. Accuracy of intra and inter-day were in the 1.0–1.6% and 1.2% for ondansetron and 0.7–2.0% and 0.3–3.8% for diphenhydramine, respectively. The limits of detection for ondansetron and diphenhydramine were 70 and 105 ng/mL, respectively, based on a signal to noise ratio of 3 and a 20 μL injection.