Gold nanoparticles (AuNPs) have shown promise for applications in the diagnosis and treatment of different diseases, including cancer. Understanding the effect of AuNPs on metabolic reprogramming in cancer cells at the single cell level is of high importance for improving the efficacy and safety. Fluorescence lifetime imaging microscopy (FLIM) of nicotinamide adenine dinucleotide (phosphate) hydrogen (NAD(P)H) as a main metabolic cofactor and an indicator of metabolic reprogramming in cancer cells enables real-time monitoring of cancer cell metabolism in response to different treatments, including AuNPs. However, NPs such as AuNPs can be a potential source of signals themselves, which provides opportunities to measure the NP internalization, but it is also important to minimize confounding effects on metabolic measurements. In this study, we detected inherent photoluminescence (PL) from the AuNPs in treated prostate cancer cells (PC-3 cell line) as well as in solution at the NAD(P)H emission wavelength. We developed an analysis approach to minimize the confounding effect of the AuNPs' PL on metabolic measurements. On the other hand, we assessed the reliability of the intracellular AuNPs' PL as an estimator of AuNP uptake. To assess if intracellular AuNPs' PL may be dependent on the exposed cell type, we performed NAD(P)H FLIM imaging of AuNP-exposed SKBR-3 breast cancer cells, where we observed a similar AuNP PL but at a much lower level compared to PC-3 cells. We proposed that this difference can be attributed to the different levels of AuNP uptake or varying intracellular microenvironments.