In the last decades, nanoparticles intended for biomedical applications have gained increased attention due to the advantages they represent among the current diagnostic and therapeutic methods. However, the translation of nanomaterials laboratory results to human therapies is limited, mainly due to incomplete characterization of nanosystem properties, before preclinical studies. In this context, this review aims to summarize the main physicochemical characterization techniques of nanoparticles in a liquid dispersion, required in their design steps; which is of utmost importance for successful applications. One of the key physicochemical parameters of nanomaterials is size. To assess nanoparticles' size, a wide revision of light scattering and microscopic techniques is reported here, some of them being also useful for determining nanomaterial morphology. The determination of nanosystem surface charge is also reported, because it is also a key parameter that will influence their interaction with biological components. In addition, the determination of nanomaterials' stability, which is important in terms of storage and use, is described. In conclusion, this review will be a useful support to find the appropriate techniques for an appropriate nanoparticle physicochemical preclinical characterization.