In the rapidly evolving field of cellular and molecular toxicology, understanding the cellular uptake of nanoparticles is crucial for assessing their potential toxicity and therapeutic applications. Nanoparticles, with their unique physicochemical properties, offer promising avenues for drug delivery, imaging, and targeted therapy. However, their interactions with biological systems can be complex and multifaceted. Flow cytometry has emerged as a powerful tool for unravelling the intricacies of cellular nanoparticle uptake. This chapter explores the principles and applications of flow cytometry in studying the cellular uptake of nanoparticles, shedding light on its significance in cellular and molecular toxicology.