Currently, in India, the National AIDS Control Organization does not recommend HIV screening for all patients attending health care facilities. The objective of study was to evaluate cost and benefits of opt-out HIV testing at a tertiary care hospital from India. This is a retrospective cohort study of patients who had undergone HIV testing. The cost for HIV testing and cost per HIV-infected patient were determined. A total of 6512 patients (66.4% men and 905 patients younger than 14 years) with mean (SD) age of 30.3 (20.7) years were tested for HIV infection during the study period. Overall, 137 (2.1%) patients tested positive for HIV infection. Total cost for performing HIV tests during study period was Indian Rupees (INR) 649,319 (US dollar [USD] 11805.8). The estimated cost per person tested was INR 99.71 (USD1.8) and cost per HIV-infected patient identified and referred to the antiretroviral therapy centre was INR 4739.55 (USD86.2). We determined a 2.1% period prevalence estimate for HIV infection. Based on cost per HIV-infected patient identified and referred to the antiretroviral therapy centre and the burden of HIV infection, it may be cost effective to perform routine opt-out screening for HIV infection in all patients attending health care facilities in developing countries like India.