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Abstract Background This study is aimed to develop a simple, effective and economic method for the UV spectrophotometric analysis of amoxicillin trihydrate in the presence of acetaminophen. The Beer–Lambert law was obeyed in the concentration range of 2–10 µg/ml for amoxicillin trihydrate, acetaminophen and combinations, in all the different pH media: pH 1.2, 6.8, 7.4 and neutral (double-distilled water). For the simultaneous equation method, the absorbance maxima of amoxicillin trihydrate was found at 228 nm, and for acetaminophen, it was found at 243 nm, after scanning the solutions in respective buffers. Result The standard curve of amoxicillin trihydrate and acetaminophen was plotted, and the correlation coefficient (R2) value was found to be in the range of 0.991–0.994 and 0.993–0.999, respectively. These two drugs were combined in a ratio of 5:3 (amoxicillin trihydrate: acetaminophen), and its absorbance maxima was discovered at 232 nm (isoabsorptive point), where its correlation coefficient was calculated from the standard curve which is in range of 0.993–0.996. The above-mentioned method was found to comply all the validation parameters as per the ICH guidelines such as accuracy, precision, linearity, LOD, LOQ, reproducibility and recovery. This method is successfully applied to estimate the combination of these two drugs in their pharmaceutical dosage forms without and interaction of their excipients. This method is based on to check the stability of amoxicillin trihydrate in different pH media in the presence of acetaminophen. Conclusion Hydrolysis of beta-lactam ring of amoxicillin trihydrate occurs in acidic pH (below 2) which causes the formation of amoxicilloic acid which may cause reduction in its microbial activity but neither shifting of wavelength nor appearance of extra peak occurred in UV spectroscopy. Although some changes of % area of amoxicillin trihydrate is observed in acidic media in HPLC method, there are no significant changes observed among the amoxicillin trihydrate solutions with acetaminophen prepared in different pH media, when using UV spectrophotometric method.