L-Amoxicillin is an antibiotic with activity against a wide range of bacterial infections. L-Amoxicillin is commonly used in antibacterial combinations when used in combination with the β-lactamase inhibitor potassium clavulanic acid. The related substances analysis method for L-Amoxicillin has been developed and validated according to the International Conference on Harmonization (ICH) guidelines, ensuring its effectiveness and accuracy in mixtures. L-Amoxicillin and some of its impurities have been further analyzed in stress testing and stability studies, providing support for optimizing its application