Biological determination of Pivampicillin hydrochloride was examined and an available method has been established. Unhydrolyzed Pivampicillin hydrochloride has no antibacterial activity, but it exhibits an antibacterial activity hydrolyzed with liberation of ampicillin by nonspecific enzymes present in blood and tissues from various species in vitro and in vivo. Using these properties, Pivampicillin hydrochloride can be determined as ampicillin by use of an agar-diffusion method with Bacillus subtilis as the assay organism.