The aim of this study was to assess the critical quality attributes of parenteral nanoemulsion formulations by measuring several physicochemical parameters and linking them to their in vitro performance, illustrating how simplistic and routinely used approaches are insufficient for understanding a potential nanomedicine. Physicochemical characterization should encompass size and size distribution through at least two orthogonal techniques, such as dynamic light scattering (DLS) and electron microscopy, with added value from analytical ultracentrifugation. In vitro toxicity assessment was performed using three different assays to determine mitochondrial activity (WST-1), membrane integrity (lactate dehydrogenase release (LDH) assay), and cell viability (propidium iodide (PI) staining). Special focus was placed on estimating appropriate incubation times for relevant results in biological investigations. All formulations had an average diameter of around 100 nm. Conclusions regarding in vitro safety were assay-dependent: LDH and PI-based assays showed good correlation, while the WST-1 assay indicated that the non-PEGylated formulation altered mitochondrial activity more significantly compared to the PEGylated ones. The study underlined that the selection of appropriate cytotoxicity assays should be based on the possible mechanism of cellular perturbation. Alternatively, different aspects of cellular toxicity should be tested. Additionally, there is a need for well-designed controls to overcome nanoparticle scattering effects and avoid potentially false high toxicity results, which was demonstrated. Combining orthogonal, well-designed physicochemical and biological assays in a standardized manner as an initial step in the reliable preclinical characterization of nanomedicines is suggested. This represents a key aspect of new methodologies in nanomedicine characterization.