ABSTRACT A comprehensive mass balance approach was developed to quantitatively determine the oxytetracycline‐free base in oxytetracycline hydrochloride. The measurement uncertainty of the reference material includes the uncertainty component introduced during the preparation process. The structural analogues of the principal components were quantitatively determined by liquid chromatography (HPLC‐DAD), moisture was determined by the Karl Fischer method, nonvolatile impurities were determined by inductively coupled plasma mass spectrometry (ICP‐MS), residual solvents were determined by headspace gas chromatography, and the content of chloride ions was determined by ion chromatography. The principal component structural analog impurities were qualitatively analyzed by HPLC‐MS, and the structural analogue impurities were quantitatively analyzed by the external standard method. The HPLC‐DAD analysis results showed that there were 17 impurities in oxytetracycline hydrochloride. Eleven out of the 17 impurities were successfully identified by HPLC‐MS. The results show that the content of structural analog impurities in oxytetracycline hydrochloride is 47.23 mg/g, the content of nonvolatile impurities is 0.18 mg/g, the content of volatile impurities is 0.89 mg/g, the content of chloride ions is 65.16 mg/g, and the water content is 89.90 mg/g. The free base content of oxytetracycline in oxytetracycline hydrochloride was 796.6 mg/g, and the expanded uncertainty was U = 9.4 mg/g ( k = 2). The method was verified by quantitative nuclear magnetic resonance (qNMR) spectroscopy. The results showed that the content of free base in oxytetracycline hydrochloride was 788.6 mg/g, and the uncertainty was U = 6.1 mg/g ( k = 2). The established mass balance method provides a strong framework for the formulation and certification of oxytetracycline reference standards and offers a reliable alternative to traditional quantitative methods. This study establishes a validated method for the characterization and quantification of oxytetracycline hydrochloride, which has potential applications in the quality control and standardization process of the pharmaceutical industry.