Advanced therapies with combined approaches of cell and nanomedicine‐based interventions are emerging. Corneal blindness with a severe form of limbal stem cell (LSC) deficiency is an example of defect with unmet clinical need. Towards cell‐based therapy, advanced enabling technologies are needed for efficient intracellular delivery of biomolecules both for in vitro disease modeling and for in vivo interventions. For this nanomedicine creates novel possibilities as light activatable polymeric nanoparticles (NPs) hold potential for controlled on‐demand cargo delivery with control of light. In this study, we used multi‐parametric surface plasmon resonance (MP‐SPR) technique in vitro for measuring NP intake in real‐time by therapeutic LSCs. Although a variety of NPs has been described, many challenges remain, and one is related to cell uptake of the formulations. Here, three different NP formulations were analyzed, one demonstrating clearly the highest cellular intake by LSCs. Importantly, data demonstrate that the labeling of NPs significantly reduce the cellular intake highlighting the importance of label‐free administration and quantification. The MP‐SPR based approach hold high potential as a powerful non‐invasive platform to be implemented in manufacturing of therapeutic cell‐ and cargo‐based interventions. For any therapy applications, validated non‐invasive and label independent NP intake measurement systems would be important.