Aim: In this work we illustrate limits and challenges associated with the use of pharmacological inhibitors to study how nanomedicines enter cells and show how such limits can be overcome. Materials & methods: We selected a panel of six common pharmacological inhibitors and a model nanoparticle-cell system. We tested eventual toxicity by measuring cell viability. We confirmed drug efficacy by measuring the uptake of control markers for the pathways involved by flow cytometry and fluorescence microscopy. Results & conclusion: We show how to optimize the use of pharmacological inhibitors and interpret the results generated. Furthermore, we demonstrate that some inhibitors cannot be used for nanomedicine studies because they lose their efficacy when serum is added, as required for nanoparticle exposure to cells.