Nef is a multifunctional protein encoded by all primate lentiviruses that modulates cell surface expression of a variety of cellular receptors and increases the infectivity of progeny virons. Here, we describe the use of bicistronic HIV-1 constructs that coexpress Nef and fluorescent proteins via an internal ribosome entry site to quantify Nef-mediated receptor modulation in virally infected cells. We also report how such proviral constructs and indicator cell lines can be used to quantify the effect of Nef on virion infectivity.