The removal and inactivation of infectious human norovirus (HuNoV) is a major focus in water purification, but the effectiveness of disinfection processes on norovirus is largely unknown owing to the lack of a readily available infectivity assay. In particular, norovirus behavior through unit processes may be over- or underestimated using current approaches for assessing HuNoV infectivity (e.g., surrogates, molecular methods). Here, we fill a critical knowledge gap by estimating inactivation data for HuNoV after exposure to UV254, a commonly used disinfection process in the water industry. Specifically, we used a PCR-based approach that accurately tracks positive-sense single-stranded RNA virus inactivation without relying on culturing methods. We first confirmed that the approach is valid with a culturable positive-sense single-stranded RNA human virus, coxsackievirus B5, by applying both qPCR- and culture-based methods to measure inactivation kinetics with UV254 treatment. We then applied the qPCR-based method to establish a UV254 inactivation curve for HuNoV (inactivation rate constant = 0.27 cm2 mJ-1). Based on a comparison with previously published data, HuNoV exhibited similar UV254 susceptibility compared with other enteric single-stranded RNA viruses (e.g., Echovirus 12, feline calicivirus) but degraded much faster than MS2 (inactivation rate constant = 0.14 cm2 mJ-1). In addition to establishing a HuNoV inactivation rate constant, we developed an approach using a single qPCR assay that can be applied to estimate HuNoV inactivation in UV254 disinfection systems.