Introduction: Genital Herpes Simplex Virus (HSV) infection is the most common infection in Acquired Immunodeficiency Syndrome (AIDS) patients, occurring in 60-90% according to World Health Organisation (WHO) reports. Early detection of HSV-2 infection, the introduction of chemotherapy, and prophylaxis improve the lifespan of AIDS patients. Aim: To detect the HSV-2 seroprevalence that is Immunoglobulin G (IgG) and Immunoglobulin M (IgM) antibodies in Human Immunodeficiency Virus (HIV) positive serum and molecular detection of HSV-2 Deoxyribonucleic Acid (DNA) in symptomatic HSV-2 Patients. Materials and Methods: The descriptive cross-sectional study was conducted in the Guntur Government Hospital with the help of the Antiretroviral Therapy (ART) Centre and Department of Dermatology, Venereology and Leprosy (DVL) in GGH, Guntur, Andhra Pradesh, India. A total of 100 blood samples from HIV positive individuals were collected and for those with symptoms on the external genitalia, a biopsy specimen from the ulcer and fluid from vesicles were taken. All the cases in the study group are on ART and without any antiviral treatment. A blood sample was collected for IgG (Calbiotech kit), IgM detection (Merilisa kit), and biopsy from genital ulcer for gene detection by realtime Polymerase Chain Reaction (PCR) (HELINI Biomolecules, Chennai). Statistical analysis was done by mean and percentages with p-value by calculating Chi-square test, Fisher’s-Exact test to know significance of association between HSV-2 and HIV. Results: Among the total 100 HIV positive patients, it was observed that IgG seroprevalence of HSV-2 in HIV positive individuals is 78%, while it is 19% for IgM. In the present study, 19 symptomatic cases showed DNA of HSV-2 in PCR, out of which, 17 were from genital ulcers and two from vesicle fluid. Conclusion: As HSV-2 is a lifelong infection, serological testing provides the best method, to estimate its prevalence even in asymptomatic individuals.