Background: Herpes simplex viruses (HSVs) are responsible for a variety of human diseases. Although lesions are usually self-limited, severe manifestations can occur, particularly in compromised hosts. Effectiveness of therapy for such infections relies upon rapid administration of appropriate antivirals which in turn creates the need to establish a prompt diagnosis and necessitates diagnostic testing that is rapid, sensitive and affordable especially for laboratories in developing countries. The specificity of tests is also crucial, since clinical manifestations of HSV are relatively nonspecific and overlap other potentially severe infections. Objectives: This study aimed at comparing the performance of two relatively affordable diagnostic assays; conventional PCR and tissue culture; in the detection of HSV in different clinical specimens. Methodology: Seventy participants were included and divided into two groups. Group I: comprised 50 patients with suspected herpetic lesions. Group II: comprised 20 subjects without any herpetic clinical manifestations. Samples from participants were tested for HSV pol gene by conventional PCR. Tissue culture was performed by inoculating the samples on Vero cell line. Results: Conventional PCR showed perfect agreement with the gold standard (κ= 1) with sensitivity, specificity, and accuracy of 100%. Tissue culture assay detected 15 (21.4%) of all positive cases showing substantial agreement with the gold standard (κ= 0.632) with sensitivity, specificity and accuracy of 57.7%, 100% and 84.29%, respectively. Conclusion: Though tissue culture has its own advantages, conventional PCR could serve as a gold standard for the diagnosis of HSV infection.