The most hazardous class of pharmaceuticals for soil and aquatic ecosystems are antibiotics, which include prescription medications and cancer treatments. Hospital effluents are usually produced by all parts of medical facilities, including hospitals. This study's specific goal was to provide a quick, affordable, and accurate analytical technique for determining the levels of amoxicillin, azithromycin, and penicillin in wastewater from Medical City, Al-Mahmudiya, and Al-Yarmouk hospitals (Iraq, Baghdad). An HPLC with a receptive ODS C18 column was used. It was equipped with UV and pulsed amperometric detectors with wavelengths of 230 nm and 210-240 nm, respectively. The correlation coefficients for each drug are greater than 0.9999, indicating that the approach is linear within the given range. It was evident that the limit of detection and limit of quantification for amoxicillin, azithromycin, and penicillin, respectively, were 0.103 µg/mL, 0.312 µg/mL, 0.069 µg/mL, and 0.211 µg/mL, and 0.086 µg/mL, 0.262 µg/mL. Ultimately, these results show that all wastewater samples from Medical City Hospital contain the three antibiotics amoxicillin, azithromycin, and penicillin. However, the Al-Mahmudiya sample had higher levels of penicillin and the Al-Yarmouk sample had lower levels of amoxicillin when compared to Medical City. The suggested method applies to a wide range of healthcare products used in a variety of effluent sources, including medical facilities and manufacturing environments. For citation: Jasim M. S. Jamur, Ala’a Abdullwahid Jasim, Wasan Abdulwahid Jasim High-performance liquid chromatography for the analysis of amoxicillin, azithromycin and penicillin in river hospital waste. ChemChemTech [Izv. Vyssh. Uchebn. Zaved. Khim. Khim. Tekhnol.]. 2026. V. 69. N 1. P. 127-134. DOI: 10.6060/ivkkt.20266901.7183.