The production of semi-synthetic beta-Iactam antibiotics such as Amoxicillin may be performed enzymatically using penicillin acylase under mild conditions. However, the thermodynamically favored hydrolysis of the antibiotic product and the acyl donor substrate needs to be minimized to use the kinetically controlled route. The addition of cosolvents such as ethylene glycol and methanol (the two best solvents identified so far for semi-synthetic beta-lactam antibiotics) can achieve this to some degree, but these additives also produce enzyme inhibition and deactivation. In this study, we compared ethylene glycol and methanol under various substrate conditions. Methanol gave a better synthesis to hydrolysis ratio, although its deactivating effects adversely affected production at lower cosolvent concentrations than ethylene glycol. This effect and its dependence on substrate concentration was further modeled and optimized. A few targets of optimization such as Amoxicillin level, the synthesis to hydrolysis ratio, or a combination, were employed. While maximum levels of Amoxicillin synthesis were achievable only at high substrate concentrations, improvements derived from cosolvents were most significant at low substrate concentrations.