The human APOBEC3 (A3) cytidine deaminases, such as APOBEC3G (A3G) and APOBEC3F (A3F), are potent inhibitors of Vif-deficient human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif (viral infectivity factor) binds A3 proteins and targets these proteins for ubiquitination and proteasomal degradation. As such, the therapeutic blockage of Vif-A3 interaction is predicted to stimulate natural antiviral activity by rescuing APOBEC expression and virion packaging. In this study, we describe a successful application of the Protein Fragment Complementation Assay (PCA) based on the enzyme TEM-1 β-lactamase to study Vif-A3 interactions. PCA is based on the interaction between two protein binding partners (e.g., Vif and A3G), which are fused to the two halves of a dissected marker protein (β-lactamase). Binding of the two partners reassembles β-lactamase and hence reconstitutes its activity. To validate our assay, we studied the effect of well-described Vif (DRMR, YRHHY) and A3G (D128K) mutations on the interaction between the two proteins. Additionally, we studied the interaction of human Vif with other members of the A3 family: A3F and APOBEC3C (A3C). Our results demonstrate the applicability of PCA as a simple and reliable technique for the assessment of Vif-A3 interactions. Furthermore, when compared with co-immunoprecipitation assays, PCA appeared to be a more sensitive technique for the quantitative assessment of Vif-A3 interactions. Thus, with our results, we conclude that PCA could be used to quantitatively study specific domains that may be involved in the interaction between Vif and APOBEC proteins.