Viral Hemorrhagic Septicemia virus (VHSv) causes one of the world's most important finfish diseases, killing >80 species across Eurasia and North America. A new and especially virulent strain (IVb) emerged in the North American Great Lakes in 2003, threatening fisheries, baitfish, and aquaculture industries. Weeks-long and costly cell culture is the OIE and USDA-APHIS approved diagnostic. A new Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR) assay that uniquely incorporates internal standards to improve accuracy and prevent false negatives was developed and evaluated for its ability to detect and quantify VHSv. Results from StaRT-PCR, SYBR® green real time qRT-PCR, and cell culture were compared, as well as the effects of potential PCR inhibitors (EDTA and high RNA). Findings show that StaRT-PCR is sensitive, detecting a single molecule, with 100% accuracy at six molecules, and had no false negatives. In comparison, false negatives ranged from 14 to 47% in SYBR®green real time qRT-PCR tests, and 47–70% with cell culture. StaRT-PCR uniquely controlled for EDTA and RNA interference. Range of VHSv quantitation by StaRT-PCR was 1.0 × 100–1.2 × 105 VHSv/106actb1molecules in wild caught fishes and 1.0 × 100–8.4 × 105 molecules in laboratory challenged specimens. In the latter experiments, muskellunge with skin lesions had significantly more viral molecules (mean = 1.9 × 104) than those without (1.1 × 103) (p < 0.04). VHSv infection was detected earlier in injection than in immersion challenged yellow perch (two versus three days), with molecule numbers in both being comparable and relatively consistent over the remaining course of the experiment. Our results show that the StaRT-PCR test accurately and reliably detects and quantifies VHSv.