Gel electrophoresis is a ubiquitous bioanalytical technique used in research laboratories to validate protein and nucleic acid samples. Polyacrylamide and agarose have been the gold standard gel materials for decades, but an alternative class of polymer has emerged with potentially superior performance. Pluronic thermal gels are water-soluble polymers that possess the unique ability to undergo a change in viscosity in response to changing temperature. Thermal gels can reversibly convert between low-viscosity liquids and high-viscosity solid gels using temperature as an adjustable parameter. The properties of thermal gels provide unmatched flexibility as a dynamic separations matrix to measure analytes ranging from small molecules to cells. This review article describes the physical and chemical properties of Pluronic thermal gels to provide a fundamental overview of polymer behavior. The performance of thermal gels is then reviewed to highlight their applications as a gel matrix for electrokinetic separations in capillary, microfluidic, and slab gel formats. The use of dynamic temperature-responsive gels in bioanalytical separations is an underexplored area of research but one that holds exciting potential to achieve performance unattainable with conventional static polymers.