HIV encodes an aspartyl protease that is activated during, or shortly after, budding of viral particles from the surface of infected cells. Protease-mediated cleavage of viral polyproteins is essential to generating infectious viruses, a process known as 'maturation' that is the target of FDA-approved antiretroviral drugs. Most assays to monitor protease activity rely on bulk analysis of millions of viruses and obscure potential heterogeneity of protease activation within individual particles. In this study we used nanoscale flow cytometry in conjunction with an engineered FRET reporter called VIral ProteasE Reporter (VIPER) to investigate heterogeneity of protease activation in individual, patient-derived viruses. We demonstrate previously unappreciated interpatient variation in HIV protease processing efficiency that impacts viral infectivity. Additionally, monitoring of protease activity in individual virions distinguishes between drug sensitivity or resistance to protease inhibitors in patient-derived samples. These findings demonstrate the feasibility of monitoring enzymatic processes using nanoscale flow cytometry and highlight the potential of this technology for translational clinical discovery, not only for viruses but also other submicron particles including exosomes, microvesicles, and bacteria.