A systematic approach to quantifying the electrode kinetics of surface-confined proteins and identifying the impact of surface heterogeneity is presented. The evaluation approach is based on analysis of individual harmonics derived from Fourier transformed large-amplitude ac voltammetry, and their peak current magnitude, Ip(nωt) versus frequency, f, relationships. Effectively, variability in the time-scale of each harmonic is expected, and advantage is taken of the fact that each individual harmonic displays a different level of sensitivity with respect to the kinetic evaluation. The data strategy protocols have been examined for the azurin Cu(II)/Cu(I) process when this metalloprotein is immobilized on gold electrodes modified alkanethiols having different chain lengths, using both pure and mixed thiol systems. Ip(nωt) versus f relationships also offer the advantage of the ability to detect and allow for the ohmic IRu drop effect and allow analyses that are independent of protein surface coverage. Estimation of an electron transfer rate is achievable from this form of analysis. However, experimentally observed waveshapes for each individual harmonic are consistently broader than that deduced theoretically on the basis of their rate constants because of kinetic and/or thermodynamic dispersion. In the mixed thiol systems, and with use of the ac method, kinetic discrimination is achieved for fast processes. This systematic study based on a model protein indicates that a more comprehensive level of evaluation of electrode kinetics can be derived from analysis of the ac harmonics available in large-amplitude ac voltammetry, by initially using Ip(nωt)-f data to evaluate the electrode kinetics followed by waveshape analysis to detect heterogeneity effects that give rise to kinetic or thermodynamic dispersion. © 2009 Elsevier B.V. All rights reserved.