A new isoelectric focusing technique has been developed that incorporates natural pH gradient formation in microfluidic channels under flowing conditions. In conjunction, a one-dimensional finite difference model has been developed that solves a system of algebraic-ordinary differential equations that describe the phenomena occurring in the system, including hydrolysis at the electrodes, buffering effects of weak acids and bases, and mass transport due to both diffusion and electrophoresis. A quantitative, noninvasive, optically based method of monitoring pH gradient formation is presented, and the experimental data generated by this method are found to be in good agreement with model predictions. In addition, the model provides a theoretical explanation for initially unexpected experimental results. Model predictions are also shown to match well with experimental results of microfluidic isoelectric focusing of a single protein species. Accounting for the nonuniform velocity profile, characteristic of pressure-driven flow in microfluidic channels, is found to improve predictions of dynamic pH changes close to the electrodes and overall time required to reach steady state, but to reduce the accuracy of dynamic pH change predictions in other regions of the channel.