Gold nanoparticles continue to generate interest for use in several biomedical applications. Recently, researchers have been focusing on exploiting their dual diagnostic/therapeutic theranostic capabilities. Before clinical translation can occur, regulatory agencies will require a greater understanding of their biodistribution and safety profiles post administration. Previously, the real-time identification and tracking of gold nanoparticles in free-flowing vasculature had not been possible without extrinsic labels such as fluorophores. Here, we present a label-free imaging approach to examine gold nanoparticle (AuNP) activity within the vasculature by utilizing multiphoton intravital microscopy. This method employs a commercially available multiphoton microscopy system to visualize the intrinsic luminescent signal produced by a multiphoton absorption-induced luminescence effect observed in single gold nanoparticles at frame rates necessary for capturing real-time blood flow. This is the first demonstration of visualizing unlabeled gold nanoparticles in an unperturbed vascular environment with frame rates fast enough to achieve particle tracking. Nanoparticle blood concentration curves were also evaluated by the tracking of gold nanoparticle flow in vasculature and verified against known pre-injection concentrations. Half-lives of these gold nanoparticle injections ranged between 67 and 140 s. This label-free imaging approach could provide important structural and functional information in real time to aid in the development and effective analysis of new metallic nanoparticles for various clinical applications in an unperturbed environment, while providing further insight into their complex uptake and clearance pathways.