Recently the Tat/rev Induced Limiting Dilution Assay, or TILDA, has been proposed as a possible alternative method to quantify the HIV-1 reservoir. TILDA estimates the frequency of latently infected cells by probing, in a limiting dilution format, the presence or inducibility of tat and rev multiply spliced HIV-1 RNA. In doing so, TILDA reduces overestimation of reservoir size compared to HIV-1 DNA measurements because multiply spliced HIV-1 RNA is less likely to be transcribed from dysfunctional genomes with replication defects. TILDA is easy to perform, requires a very low input number of cells and has a fast turnaround time, making it ideal for use in clinical settings. Here we describe the execution of TILDA with particular emphasis on cell preparation and the limiting dilution scheme.