In the vicinity of neutral pH, nicotine is equilibrated between unprotonated and monoprotonated states. The equilibrium is best described by the acid dissociation constant (Ka) and its logarithmic function known as pK_a which in non-aqueous solution is termed p_sK_a. E-cigarette formulations are predominately non-aqueous, typically composed of glycerol, 1,2-propanediol, water and (S)-nicotine, and the p_sK_a of nicotine is influenced by the formulations’ concentration, solvent composition and temperature. The objective of this study was to determine the p_sK_a of nicotine in a typical e-cigarette formulation and from that value calculate the distribution between the two states of nicotine. Circular dichroism spectra of (S)-nicotine change with pH, reflecting transitions between unprotonated and monoprotonated forms. Through the addition of acid and alkali solutions we constructed titration curves at 20 °C over the pH range 4-10, whose inflection points yielded the p_sK_a value for nicotine under the specific ionic strength, co-solvent and temperature conditions of the (S)-nicotine/glycerol/water system. The concentration of (S)-nicotine was increased stepwise from 30 µg/mL to 3 mg/mL in each titration curve together with a concomitant reduction in cell path length from 10 to 0.1 mm. From the five p_sK_a values obtained, the p_sK_a value was inferred for 30 mg/mL (a typical e-cigarette formulation level). The p_sK_a at 30 mg/mL in 9% water balanced with glycerol at 20 °C was estimated as 7.24. From this value, using the Henderson-Hasselbalch equation, the proportions of unprotonated and monoprotonated nicotine may be calculated with reference to the pHs (the pH in co-solvent) of the formulation. The methodology described here is advanced as a robust approach for estimating the proportions of the two forms of nicotine that predominate in electronic cigarette formulations.