The clinical utility of HIV-1 resistance testing is dependent upon accurate interpretation and application of results. The development of clinical cut-offs (CCOs) for most HIV antiretroviral drugs assessed by the vircoTYPE HIV-1 resistance test has been described previously. Updated CCOs based on new methodology and new data from clinical cohorts and pivotal clinical studies are presented in this communication. Data for analysis included the original records for CCO derivation from eight clinical trials and two cohort studies plus new records from the clinical cohorts and from the TITAN, POWER, and DUET clinical studies. Drug-specific linear regression models were developed to describe the relationship between baseline characteristics (phenotypic resistance as estimated by virtualPhenotype-LM using methods revised recently for handling mixed viral sequences; viral load; and treatment history), new treatment regimen, and 8-week virologic outcome. The clinical cut-offs were defined as the estimated phenotypic resistance levels (fold change, FC) associated with a 20% and 80% loss of drug activity. The development dataset included 6550 records with an additional 2299 reserved for validation. The updated, v.4.2 CCOs were generally close to the v4.1 values, with a trend observed toward marginally higher cut-offs for the NRTIs. These results suggest that the updated CCOs provide a relevant tool for estimating the contribution to virological response of individual antiviral drugs in antiretroviral drug combinations as used currently in clinical practice.