The use of nanomedicines for cancer treatment holds a great potential due to their improved efficacy and safety. During the nanomedicine preclinical in vitro evaluation stage, these are mainly tested on cell culture monolayers. However, these 2D models are an unrealistic representation of the in vivo tumors, leading to an inaccurate screening of the candidate formulations. To address this problem, spheroids are emerging as an additional tool to validate the efficacy of new therapeutics due to the ability of these 3D in vitro cancer models to mimic the key features displayed by in vivo solid tumors. In this review, the application of spheroids for the evaluation of nanomedicines’ physicochemical properties and therapeutic efficacy is discussed.