Polyacrylamide gel electrophoresis (PAGE) is a ubiquitous technique used in biochemical research laboratories to characterize protein samples. Despite its popularity, PAGE is relatively slow and provides limited separation resolution, especially for native proteins. This report describes the development of a microfluidic thermal gel transient isotachophoresis (TG-tITP) method to rapidly separate native proteins with high resolution. Thermal gels were employed as a separations matrix because of their unique ability to change viscosity in response to temperature. Proteins (6–464 kDa) were added into thermal gel and loaded into a microfluidic device. Electrolyte optimization was conducted to achieve robust tITP to isotachophoretically preconcentrate proteins and then electrophoretically separate them. Electropherograms were collected through both time and distance to enable both small and large proteins to be measured within a single analysis. The effects of temperature were evaluated and found to exhibit a pronounced effect on the separation. Temperature gradients were then employed to alter thermal gel viscosity over time to maximize separation resolution between proteins. The results herein demonstrate how gradient TG-tITP achieves rapid, high-resolution separations of native proteins over a wide mass range while requiring significantly less protein loading than PAGE and providing faster analysis times.