Lab-on-a-chip electrophoresis is becoming increasingly useful for protein analysis, thanks to recent developments in this field. This review is an update of the review we published at the start of 2008 [Peng, Y., Pallandre, A., Tran, N. T., Taverna, M., Electrophoresis 2008, 29, 156-177]. The superiority of polymers for the manufacture of analytical microchips has been confirmed. This trend implies several modifications to the processes previously used with glass/silicon chips and requires a better understanding of the interfacial phenomena of these materials. Significant progress in chip-based techniques for protein analysis has been made in the last 2 years. In addition to advances in traditional electrokinetic modes, counter-flow gradient focusing techniques have emerged as useful methods not only for separation, but also for the online preconcentration of samples. This review, with more than 175 references, presents recent advances and novel strategies for EOF measurement, surface treatment, sample pretreatment, detection and innovations relating to the different modes of separation.