Objective To establish a reverse-phase high performance liquid chromatography (RP-HPLC) gradient elution method for the simultaneous determination of ephedrine, sulfamethoxazole and dexamethasone acetate in nasal drops. Methods The samples were separated on a Agilent Eclipse XDB-C18 (250 mm×4.6 mm, 5 μm) by mobile phase of methanol (A) and 0.1% phosphonic acid solution (B) with gradient elution. The program of gradient elution was as follows: The volume ratio of mobile phase A:B was 30:70 between 0 min and 10 min; the volume ratio of mobile phase A:B was 70:30 between 10 min and 18 min; the volume ratio of mobile phase A:B was 30:70 between 18 min and 20 min. The flow rate was 1.0 mL/min and the program of UV gradient absorbance detection was as follows: 0-5 min, at 220 nm; 5-8 min, at 310 nm; 8-20 min, at 240 nm. The column temperature was 30℃ and the sample volume was 10 μL. Results The linear concentration ranges of the calibration curves for ephedrine, sulfamethoxazole and dexamethasone acetate were 0.083-0.667, 0.417-3.333 and 0.003-0.027 g/L, and the coefficient of correlation for ephedrine, sulfamethoxazole and dexamethasone acetate were 0.999 7 (n=5), 0.999 8 (n=5) and 0.999 5 (n=5), respectively. The average recoveries for ephedrine, sulfamethoxazole and dexamethasone acetate were (100.2±1.6)% with RSD of 1.61%, (100.4±1.3)% with RSD of 1.29%, and (99.8± 1.4)% with RSD of 1.74%, respectively. The average content for ephedrine, sulfamethoxazole and dexamethasone acetate were (97.2±3.1)% with RSD of 3.06%, (98.6±1.1)% with RSD of 1.13%, and (97.8±1.2)% with RSD of 1.23%, respectively. Conclusions The RP-HPLC gradient elution method is rapid, sensitive, precise and specific. It could be used to control the quality of dexamethasone acetate, sulfamethoxazole and ephedrine nasal drops.