A simple, cost-effective, and greener HPLC method was investigated to determine 14 β-lactams. To achieve this, various strategies were implemented such as the use of an aqueous mobile phase, ionic liquids, or an alternative organic solvent. Preliminary experiments were conducted using "one-factor-at-atime" to identify the significant factors affecting chromatographic performance. The experimental design (central composite design) was then applied to thoroughly examine the effects of the key factors and exploit them to achieve optimal HPLC condition. The developed method was less hazardous and improved the retention and peak shape, compared with other conventional methods. The optimized assay condition was validated according to the ICH guidelines and effectively applied for the quantification of some β-lactams in various formulations. These results imply that this novel HPLC method can be employed for routine analyses of some β-lactams in pure drug and commercial formulations.