A new triplex real-time polymerase chain reaction (PCR) assay for Herpes simplex virus (HSV) (artus HSV-1/2 TM PCR kit, QIAGEN) was evaluated. This assay simultaneously uses three differently labeled probes targeted to HSV-1 (FAM), HSV-2 (NED), and to the manufacturer's Internal Control (VIC). HSV-1/2 typing capability and quantitation accuracy were determined using HSV stocks and quality control panels. Performance in routine clinical testing was compared with a nested HSV-1/2 PCR assay. Dilution series and quality control panel testing revealed an approximately 10-fold higher HSV-2 sensitivity in real-time PCR compared with an in-house nested PCR assay. The sensitivity for HSV-1 was comparable in both assays. All HSV-positive proficiency panel samples (n = 21) and virus stocks were typed correctly as HSV-1 or HSV-2 using real-time PCR. Quantitation correlated well with reference values (HSV-1, r = 0.98; HSV-2, r = 0.88), and 95% detection limits were determined as 9.4 HSV-2 copies/reaction and 18 HSV-1 copies/reaction. Based on C(t) values, the mean intra-assay coefficient of variation was 1%, whereas the interassay coefficient of variations were 2.7% and 2.5% for HSV-1 and -2, respectively. Testing of 309 clinical samples resulted in 100% specificity and 97% sensitivity. In conclusion, the artus HSV-1/2 TM PCR kit represents an excellent tool for the detection and differentiation of HSV-1 and -2 in clinical samples.