The realization of an integrated lab-on-a-chip system for the analysis of proteins has been hindered by several non-trivial challenges. Among these challenges are that the reagents required for one process can often be incompatible with a downstream process. One highly researched technique is the integration of electrophoretic separations of proteins and analysis of the proteins by mass spectrometry. Sodium dodecylsulfate (SDS) is often needed to solubilize hydrophobic proteins and provides a constant charge-to-mass ratio resulting in a purely size-based separation. However, a drawback to this system is that SDS has been shown to interfere with mass spectrometry. In this study, an acid-labile surfactant (ALS) was investigated as a replacement for SDS in the electrophoretic separation of proteins on a microfluidic chip. ALS is advantageous as it degrades at low pH (pH < 3), and therefore may facilitate the integration of electrophoresis with mass spectrometry. We show the separation of proteins between 18 kDa and 116 kDa on a glass microfluidic chip dynamically coated with poly-N-hydroxyethylacrylamide. The proteins were labeled with the Py-1 dye, which does not alter the charge of the protein, and detected using laser-induced fluorescence. Multiple field strengths and SDS or ALS concentrations were tested to determine their effects on protein separation. Separation efficiency and migration times of a protein ladder are compared using SDS and ALS as the surfactants in the running buffer. While SDS provides a higher separation efficiency, the separation achieved by ALS appears to be sufficient to be used as a sized-based separation method prior to mass spectrometry. The separation efficiencies for proteins with ALS as the surfactant maintained peak efficiencies between 10⁴ and 10⁵ plates / meter. This demonstration of an acid-labile surfactant as a replacement for SDS in the size-based separation of protein will aid the integration of microchip electrophoresis with mass spectrometry.