Gold nanoparticles (GNPs) have become essential tools used in nanobiotechnology due to their tunable plasmonic properties and low toxicity in biological samples. Among the available approaches for imaging GNPs internalized by cells, hyperspectral techniques stand out due to their ability to simultaneously image and perform spectral analysis of GNPs. Here, we present a study utilizing a recently introduced hyperspectral imaging technique, live-cell PWS, for the imaging, tracking, and spectral analysis of GNPs in live cancer cells. Using principal components analysis, the extracellular or intracellular localization of the GNPs can be determined without the use of exogenous labels. This technique uses wide-field white light, assuring minimal toxicity and suitable signal-to-noise ratio for spectral and temporal resolution of backscattered signal from GNPs and local cellular structures. The application of live-cell PWS introduced here could make a great impact in nanomedicine and nanotechnology by giving new insights into GNP internalization and intracellular trafficking.