Background: The main goal of process-related substances investigations is to understand how impurities should be controlled during the synthetic or purification process to produce a high-quality drug substance. The simultaneous determination of related substances is a very important factor in the quality control of API and pharmaceutical dosage forms. Methods: A new HPLC method was developed for the simultaneous analysis of Ipratropium bromide and its seven related substances. The separation was performed on a waters HPLC ZORBAX Eclipse XDB- C8 column (150 x 4.6 mm, 3.5 mu m). The gradient method used mobile phases containing methanol, acetonitrile, trifluoroacetic acid (TFA), water: A: (methanol, acetonitrile, TFA, 500:500:0.3 v/v/v) and B (0.3% TFA in water v/v). The gradient elution conditions were as follows: 0-32.0 min, linear from 10% A to 35% A; 32.0-36.0 min isocratic 35% A; 36.0-42.0 min linear from 35% to 10%A. The wavelength by UV detector was 220 nm and the flow rate was 1.2 mL/min. The injection volume of sample was 20 mu L, and the column temperature was 30 degrees C. The proposed RP-HPLC method was applied for the determination of seven related substances. Results: All standard curves obtained exhibited good linear regression (r > 0.9939) within the tested range. The average recovery rates were in the range of 98.2-102.0% and RSD was less than 2.9% (n = 3). The process-related impurities were not observed in different batches (<0.025% purity). Conclusion: The method was applied to determine 7 process-related substances in ipratropium bromide and proved to be robust, linear, repeatable, sensitive, selective and easy to perform. The results are valuable for quality control in the manufacturing of ipratropium bromide.