CZE was employed to determine acid–base dissociation constants (pKa) of ionogenic groups of azahelicenes in methanol (MeOH). Azahelicenes are unique 3-D aromatic systems, which consist of ortho-fused benzene/pyridine units and exhibit helical chirality. The pKa values of pyridinium groups of the studied azahelicenes were determined from the dependence of their effective electrophoretic mobility on pH by a nonlinear regression analysis. The effective mobilities of azahelicenes were determined by CZE at pH range between 2.1 and 10.5. Thermodynamic pKa values of monobasic 1-azahelicene and 2-azahelicene in MeOH were determined to be 4.94 ± 0.05 and 5.68 ± 0.05, respectively, and pKa values of dibasic 1,14-diazahelicene were found to be equal to 7.56 ± 0.38 and 8.85 ± 0.26. From these values, the aqueous pKa of these compounds was estimated.