One of the major barriers to the successful design and implementation of human immunodeficiency virus (HIV) curative strategies is the limited ability to sensitively, specifically, and precisely quantify and characterize the whole-body burden of replication-competent HIV in individuals on effective antiretroviral therapy. Here, we review the development and validation of assays that directly and indirectly measure the size and distribution of the reservoir in blood and tissues. We also discuss the role that treatment interruptions will have in validating these assays and ultimately as a “proof of cure.”