For amphotericß-lactam antibiotics, the acid dissociation constant (pKa) is a fundamental parameter to characterize physicochemical and biochemical properties of antibiotics and to predict persistence and removal of drugs. pKaof piperacillin (PIP) is determined by potentiometric titration with a glass electrode. Electrospray ionization mass spectrometry (ESI-MS) is creatively applied to verify the reasonable pKavalue at every dissociation step. Two microscopic pKavalues (3.37 ± 0.06 and 8.96 ± 0.10) are identified and attributed to the direct dissociation of the carboxylic acid functional group and one secondary amide group, respectively. Different from otherß-lactam antibiotics, PIP presents a dissociation pattern where direct dissociation is involved instead of protonation dissociation. Moreover, the degradation tendency of PIP in an alkaline solution may alter the dissociation pattern or dismiss the corresponding pKaof the amphotericß-lactam antibiotics. This work offers a reliable determination of the acid dissociation constant of PIP and a clear interpretation of the effect of stability of antibiotics on the dissociation process.