Nanomedicine has emerged as a promising strategy to address some of the limitations of traditional biomedical sensing, imaging and therapy modalities. Its applicability and efficacy are, in part, hindered by the difficulty in both controllably delivering nanoparticles to specific regions and accurately monitoring them in tissue. Gold nanoparticles are among the most extensively used inorganic nanoparticles which benefit from high biocompatibility, flexible functionalization, strong and tunable resonant absorption, and production scalability. Moreover, their capability to enhance optical fields at their plasmon resonance enables local boosting of non-linear optical processes, which are otherwise very inefficient. In particular, two-photon induced luminescence (TPL) in gold offers high signal specificity for monitoring gold nanoparticles in a biological environment. In this article, we demonstrate that TPL microscopy provides a robust sub-micron-resolution technique able to quantify accumulated gold nanorods (GNRs) both in cells and in tissues. First, the temporal accumulation of GNRs with two different surface chemistries was measured in 786-O cells during the first 24 hours of incubation, and at different nanoparticle concentrations. Subsequently, GNR accumulation in mice, 6 h and 24 hours after tail vein injection, was quantified by TPL microscopy in biopsied tissue from kidney, spleen, liver and clear cell renal cell carcinoma (ccRCC) tumors, in good agreement with inductively coupled mass spectroscopy. Our data suggest that TPL microscopy stands as a powerful tool to understand and quantify the delivery mechanisms of gold nanoparticles, highly relevant to the development of future theranostic medicines.