A simple, cost-effective and greener high-performance liquid chromatography (HPLC) method was investigated to determine fourteen β-lactams. In this study, some strategies were implemented to achieve a greening HPLC method such as the use of aqueous mobile phase, alternative organic solvent or ionic liquids. Firstly, the preliminary screening step was conducted using one-factor-at-a-time approach to identify the significant factors affecting the chromatographic performance. Then the experimental design (central composite design) was applied to examine in depth the effects of the most important factors on the selected chromatographic responses as well as to find out the optimal HPLC condition. Finally, the chromatographic analysis was achieved on a C8 column at 35oC, mobile phase including ethanol and 25 mmol/ L ammonium acetate pH 5.0 (6 : 94, v/v) with the flow rate of 1.5 mL/ min, and the detector wavelength at 210 nm. This method was not only found to be less hazardous (the use of greener organic solvent, the reduction in organic solvent consumption) but also turned out to be superior in terms of retention time and peak shape in comparison with other official methods publised in the pharmacopoeias. The optimized assay condition was validated according to the International Conference on Harmonization guidelines. The results showed good linearity (R2>0.9991) over the assayed concentration range, and demonstrated good precision as well as accuracy for all surveyed compounds. The proposed method was also effectively applied for the quantitation of ampicillin, sulbactam, cefroxadine, amoxicillin, and ertapenem in various formulations. These results indicate that the developed HPLC method could be employed for routine analysis of some β-lactams in pure drug and commercial formulations.