A method for the determination of both isoprenaline hydrochloride injection and epinephrine hydrochloride injection and their related substances was established.An HPLC was performed on a column of Welch Materials C18 (150 mm × 4.6 mm, 5 μm) with the mobile phase of methanol-acetonitrile-sodium heptanesulfonate solution (1:1:8) at detection wavelength of 280 nm.For isoprenaline hydrochloride, the calibration curve was linear (r = 0.9999) over the range of 10-40 μg/mL-1, the average recovery was 100.5% with RSD 0.76%, and the limit of detection was 40.66 ng/mL-1.For epinephrine hydrochloride, the calibration curve was linear (r=0.9996) over the range of 60-240 μg/mL-1, the average recovery was 100.2% with RSD 0.60%, and the limit of detection was 44.12 ng/mL-1.This method was simple, rapid, accurate, high sensitive and good reproducible, which could be applied in the determination of both isoprenaline hydrochloride injection and epinephrine hydrochloride injection and their related substances.