Ampicillin belongs to the class of beta-lactam antibiotics, which corresponds the most prescribed antibiotics in medicine. This justifies the importance of elucidating the effect of several variables for the synthesis process (pH, temperature, co-solvent) on the behavior of the compounds involved in the enzymatic reaction. This paper aims to present new ampicillin solubility data in aqueous medium and the characterization of the solid phase of the antibiotic. In the phase equilibrium experiments it was considered the effect of pH (3 up to 7.5), temperature (283.15 K up to 298.15 K) and the ethanol concentration in the aqueous solution (varying between 0 and 70 wt% ethanol). The solubility curves deviate from U-shape solubility curves with respect to pH in the range of 0e70 wt%. This is due to ampicillin anhydrous becomes trihydrated molecule in the solid phase at alkaline pH region, changing the antibiotic properties significantly. This change in crystalline structure was confirmed by characterization of antibiotic solid phase carried out from thermogravimetric (TGA), Fourier Transform Infrared (FTIR) spectroscopy, Scanning Electron Microscopy (SEM) and X-ray Diffraction (XRD) analyses. Dissociation constants have also been measured at the studied values of temperature and ethanol composition, using potentiometric titration. Finally, a thermodynamic model considering the ideal solution was applied to describe mathematically the solubility curves measured close to experimental uncertainties