CD4+ T lymphocytes are currently the most common surrogate marker indicating immune status and disease progression with HIV infection. The cost of monitoring disease progression and response to therapy is still prohibitively expensive. Flow cytometry is the gold standard for the estimation of CD4+, but the high initial investment for this technology and expensive reagents makes it unaffordable for developing countries like India. We evaluated the Coulter cytosphere assay for quantifying CD4+ T lymphocytes in comparison with the standard method, flow cytometry, in 122 HIV-infected individuals. The correlation coefficient of the cytosphere assay compared with that of flow cytometry for CD4+ T lymphocytes was 0.97 (P< 0.0001), with a confidence interval of 0.95 to 0.98. The sensitivity, specificity, positive predictive value, and negative predictive value of the cytosphere assay in enumerating absolute CD4+ T-lymphocyte counts of less than 200/microL were 94.9%, 96.4%, 92.5%, and 97.6%, respectively. This is a simple inexpensive method and has a strong correlation with flow cytometry. Hence, the cytosphere assay can be an alternate to flow cytometry for the estimation of CD4+ T-lymphocyte counts, especially in resource-poor settings of developing countries, for monitoring HIV progression and response to therapy.