Nanoparticle technologies have tremendous potential in clinical medicine. To fully realize this potential, one must further understand how nanoparticles interact with biological systems. Typically, reporters that are conjugated to nanoparticles during synthesis are used to monitor the nanoparticles in biological systems. Unfortunately, conjugating reporters to nanoparticles complicates the synthesis and the reporter itself may alter the nanoparticle properties. To address these challenges, a copper-catalyzed azide–alkyne cycloaddition strategy has been developed to functionalize nanoparticles with fluorescent reporters after they have been delivered to biological systems. Using polyamidoamine dendrimers as model nanoparticles, the utility of this strategy is shown in several biological systems including a cancer cell model, primary immune cells, and a murine model of inflammation. This reporter strategy simplifies the synthesis without sacrificing the ability to monitor the nanoparticle conjugates. It is expected that this bioorthogonal reporter strategy can be used to understand nanoparticle interactions in biological systems, which will facilitate the translation of these technologies to the clinics.