Nanoparticles (NPs) can offer many advantages over traditional drug design and delivery, as well as toward medical diagnostics. As with any medical device or pharmaceutical drug intended to be used for in vivo biomedical applications, NPs must be sterile. However, very little is known regarding the effect of sterilization methods on the intrinsic properties and stability of NPs. Herein a detailed analysis of physicochemical properties of two types of AuNPs upon sterilization by means of five different techniques is reported. In addition, cell viability and production of reactive oxygen species are studied. The results indicate that sterilization by ethylene oxide seems to be the most appropriate technique for both types of NPs. It is concluded that it is crucial to test several methods in order to establish the specific type of sterilization to be performed for each particular NP.