Protamines, sperm-specific nuclear proteins, are essential for sperm chromatin condensation and DNA stabilization. They are small, highly basic, and rich in disulfide bonds. Under reducing conditions, protamines, along with other basic proteins, are soluble in acid solutions. Because of their small and similar molecular weights, SDS-PAGE cannot resolve protamine 1 and protamine 2 well. Urea-acid gel electrophoresis separates proteins based on the level of the positive charge and is thus a suitable method for resolving protamines 1 and 2. Here, we describe the commonly used protamine extraction method and the Urea-acid gel electrophoresis for assessment of protamine 1/protamine 2 ratio.