Our aim was to evaluate whether betaine has a protective effect during exposure of the human oral mucosa in vivo to sodium lauryl sulfate (SLS) or cocoamidopropylbetaine (CAPB) as measured with a multifrequency electrical impedance spectrometer (EI). Both detergents were used at the concentration of 2.0% w/v with and without 4.0% w/v betaine in distilled water in 20 volunteers, and 0.5% and 1.0% w/v SLS combined with 4.0% w/v betaine in 5 volunteers. EI measurements were taken before application of the test solutions, after their removal, and every 15 min up to 45 min. Both 0.5% and 1% SLS solutions showed a significant reduction in 3 of the 4 indices, indicating mucosal irritation after the 15-min exposure (P < 0.05), whereas 2% SLS did so in all 4 indices (P < 0.001). Betaine had no effect on the detergent-induced decline with either the 2% or the 0.5% SLS solutions. However, when combined with the 1% SLS solution, betaine significantly (P < 0.05) reduced mucosal irritation by abolishing decreases in indices MIX (magnitude index) and IMIX (imaginary part index) and lowering it for PIX (phase index). The 2% CAPB solution showed a significant (P < 0.05) reduction in all 4 indices after the 15-min exposure, but the effect was significantly weaker than that of 2% SLS (P < 0.05). Betaine did not reduce the irritating effect of 2% CAPB. These findings can be used in the development of less irritating products for oral health care.