Diagnosis of herpes simplex encephalitis (HSE) is based on the detection of herpes simplex virus (HSV) DNA in patients’ CSF samples. HSV DNA quantitation has the potential for estimating the effects of antiviral therapy. The aim of this study was to diagnose HSV DNA in HSE suspected patients and the quantitative analysis of its genome using real-time PCR to assess the value of the viral load in the course of antiviral treatment. The CSF samples were collected from 236 consecutive HSE suspected patients from November 2004 to May 2008. Upon DNA extraction, the samples were analyzed by Real-Time PCR assay. A set of primers amplified a common sequence of HSV glycoprotein B gene. The copy numbers of unknown samples were expressed via a standard curve drawn with a known amount of amplified cloned plasmid. Of the 236 samples, 137 (58%) came from males and 99 (42%) from females. The HSV genome was detected in 22 (9.3%) patients by PCR, 13 males/9 females. Serial CSF samples were available from 10 of the 22 patients. The range of the HSV DNA copy numbers in the clinical samples ranged from 2.5 × 102 to 1.7 × 106 copies/mL of CSF. Quantitative PCR results can be helpful in evaluating the efficacy of antiviral therapy in the above-mentioned patients. There is an association between the initial viral load and the duration of treatment course.