Schiff base derivatives of benzothiazoles were synthesized using substituted 2-aminobenzothiazoles and different substituted benzaldehydes. The structures of the synthesized Schiff bases were determined using Fourier transform infrared spectroscopy, H-1 NMR, C-13 NMR, and elemental analyses. The acid dissociation behavior, which was visible in UV-visible absorption spectra, of the 10 novel benzothiazole Schiff bases was studied. The acidity constants (pKa), the structures, and the protonation mechanisms of the studied molecules were obtained. The substituent effect on both phenyl rings of these synthesized benzothiazole Schiff base derivatives was observed, and the experimentally calculated deprotonation values were between 11.47 and 8.91, and the protonation values were between 4.54 and 3.52. Also, the protonation sites of synthesized benzothiazole Schiff base were investigated using H-1 NMR and FT-IR. The theoretical calculations were performed with the CACHE semiempirical method (AM1, PM3, and PM5). The best correlation between the experimental and theoretical pKa and acidity values of the compounds was obtained using the PM5 calculation method.