Spatial and spectral information of a nanocarrier and its payload is crucial for the advancement of luminescence-based imaging, disease detection, and treatment in complex biological environment. However, it remains challenging to track and quantify the delivery and localization of drugs lacking inherent fluorescence. It is demonstrated that sub 30 nm phospholipid-stabilized nanoparticles can be detected and quantified using hyperspectral transmitted light microscopy without using a fluorophore. In two proposed model systems, phospholipid-passivated carbon nanoparticles incorporate the drug in either free form or as a lipid-based prodrug. Following a rigorous characterization of these nanoparticles, in vitro toxicities via loss in cell growth density and mitochondrial respiration is studied in MCF-7 breast cancer cells. Furthermore, a detailed inhibitor based study reveals that these particles are internalized based on a clathrin-mediated pathway, irrespective of the choice of drug formulation. Hyperspectral imaging is performed to obtain the colocalization of carbon nanoparticles and drug molecules intracellularly and can successfully be tracked while therapeutic release is quantified in 3D space. The present work demonstrates that nanoparticles and therapeutic agents can be mapped and measured simultaneously barring the requirement of a dye, thus providing new avenues in the spatiotemporal characterization and synchronous detection and quantification of payload and carrier.