Despite the beneficial properties and outstanding potential of hydrogels for biomedical applications, several unmet challenges must be overcome, especially regarding to their known sensitivity to conventional sterilization methods. It is crucial for any biomaterial to withstand an efficient sterilization to obtain approval from regulatory organizations and to safely proceed to clinical trials. Sterility assurance minimizes the incidence of medical device-related infections, which still constitute a major concern in health care. In this review, we provide a detailed and comprehensive description of the published work from the past decade regarding the effects of sterilization on different types of hydrogels for biomedical applications. Advances in hydrogel production methods with simultaneous sterilization are also reported. Terminal sterilization methods can induce negative or positive effects on several material properties (e.g., aspect, size, color, chemical structure, mechanical integrity, and biocompatibility). Due to the complexity of factors involved (e.g., material properties, drug stability, sterilization conditions, and parameters), it is important to note the virtual impossibility of predicting the outcome of sterilization methods to determine a set of universal rules. Each system requires case-by-case testing to select the most suitable, effective method that allows for the main properties to remain unaltered. The impact of sterilization methods on the intrinsic properties of these systems is understudied, and further research is needed. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2472-2492, 2018.