To establish a competent method of microbiological assay for bacampicillin, one of new semi-synthetic derivatives of ${eta}-lactam$ antibiotics, a comparison was carried out between different conditions for hydrolysis of bacampicillin into ampicillin which was then subjected to cylinder plate method. The results showed that the use of carboxylic ester hydrolase in vitro as a pretreatment of it in either pH value 6 or 8 was feasible and that the cylinder plate method with Sarcina lutea was adequate for potency estimation.