Background: Affordable, easy-to-use, and reliable CD4+ T lymphocyte enumeration systems are needed in resource-constrained settings to monitor HIV. - Methods: A simple image cytometer was used to count fluorescently labeled CD4+ T and CD8+ T lymphocytes from CD3 immunomagnetically selected cells on blood specimens of 460 HIV-1-infected patients in Siriraj Hospital, Bangkok, Thailand. Results were compared with flow cytometry (FCM). - Results: CD4+ T lymphocyte counts by image cytometer were comparable (R ≥ 0.97) with those by the FACSCount and the FACScan with a bias of 7.3 and 9.1%, respectively. At very low CD4+ T lymphocyte counts (≤50/μl) some over-count outliers were observed by the FACScan and image cytometer when compared with the FACSCount. For CD8 enumeration, the image cytometer showed a good correlation (R = 0.96) and a consistent undercount (~17%) when compared with the FACSCount. - Conclusions: Evaluation of the image cytometer for CD4 and CD8 enumeration demonstrated comparable results with FCM on a population of HIV-1-infected patients. The image cytometer is a good alternative method for point-of-care settings in resource-constrained countries.