BACKGROUND: Plasma protein binding (PPB) is a critical factor in drug therapy and understanding free compound exposure across preclinical and clinical species is vital for developing new antibiotics. Optimizing beta-lactam dosing based on unbound drug concentrations has garnered significant interest, yet comprehensive data on how inter-species differences in protein binding affect the attainment of targeted unbound concentrations remain sparse. METHODS: This study aimed to examine the protein binding of three beta-lactams: cefiderocol, ceftriaxone, and temocillin using human, bovine, and rat plasma. Total and unbound beta-lactam concentrations were measured through ultrafiltration. An interspecies comparison of PPB was conducted to evaluate variability in protein binding across the different species. RESULTS: The findings revealed that PPB was highest in human plasma for all three beta-lactam antibiotics tested. In rat plasma, PPB was higher for cefiderocol and ceftriaxone compared to bovine plasma, while bovine plasma exhibited higher PPB for temocillin compared to rat plasma. CONCLUSION: Significant variability in protein binding was observed among and between different species for the tested drugs. The study highlights substantial interspecies differences in the plasma protein binding of cefiderocol, ceftriaxone, and temocillin. Our findings indicate the need for careful consideration of species-specific PPB in the optimization of beta-lactam dosing and the development of new pharmaceuticals.