Amoxicillin trihydrate is that the 4-hydroxy analog of ampicillin significantly used for the infections caused by pathogenic bacteria. The present investigation states that the study of antibacterial activity and spectrophotometric analysis with both expired and non-expired medication. The microorganisms were used for the antibacterial activity which was done by agar well diffusion method. Escherichia coli, Pseudomonas fluorescence bacteria were given the high cytotoxic effect in both expired and non-expired drugs of amoxicillin trihydrate were Streptococci aureus given the less inhibition zone once compare with gram -positive bacterium. The easy and precise spectrophotometric method was done to develop and validate the estimation of both expired and non-expired amoxicillin trihydrate in the dosage of 250mg.The calibration curves were formed in the concentration range of 10-80μg/ml. The correlation coefficient, linearity, LOD, LOQ and RSD values were found for both expired and non-expired drugs.