Hydrogels based on stimuli-responsive polymers can change their characteristics in response to small variations in environmental conditions, such as temperature, pH, and ionic strength, among others. In the case of some routes of administration, such as ophthalmic and parenteral, the formulations must meet specific requirements, namely sterility. Therefore, it is essential to study the effect of the sterilization method on the integrity of smart gel systems. Thus, this work aimed to study the effect of steam sterilization (121 °C, 15 min) on the properties of hydrogels based on the following stimuli-responsive polymers: Carbopol®940, Pluronic®F-127, and sodium alginate. The properties of the prepared hydrogels—pH, texture, rheological behavior, and sol-gel phase transition—were evaluated to compare and identify the differences between sterilized and non-sterilized hydrogels. The influence of steam sterilization on physicochemical stability was also investigated by Fourier-transform infrared spectroscopy and differential scanning calorimetry. The results of this study showed that the Carbopol®940 hydrogel was the one that suffered fewer changes in the studied properties after sterilization. By contrast, sterilization was found to cause slight changes in the Pluronic®F-127 hydrogel regarding gelation temperature/time, as well as a considerable decrease in the viscosity of the sodium alginate hydrogel. There were no considerable differences in the chemical and physical characteristics of the hydrogels after steam sterilization. It is possible to conclude that steam sterilization is suitable for Carbopol®940 hydrogels. Contrarily, this technique does not seem adequate for the sterilization of alginate or Pluronic®F-127 hydrogels, as it could considerably alter their properties.