We analyzed the impact of HIV viral load on the performance of a limiting antigen avidity enzyme immunoassay (LAg-Avidity assay) and determined if this assay could be used to identify viral breakthrough. Three groups of samples were tested: (1) 18 individuals (30 samples) previously identified as elite suppressors; (2) 18 individuals (72 samples) who were continually suppressed on antiretroviral treatment (ART) with 1 sample before and 2-6 samples (one/year) after ART initiation; and (3) 20 individuals (179 samples) on ART who had evidence of viral breakthrough (>400 copies/ml) with subsequent viral suppression. Elite suppressors had the lowest LAg-Avidity assay values. Among those who were continually suppressed on ART, 83% (15/18) had LAg-Avidity assay values that decreased over time. Although the LAg-Avidity assay on a single sample cannot identify when a viral breakthrough occurs, paired longitudinal samples could identify viral breakthrough (sensitivity: 65%, specificity: 84%).