An accurate measure of infectious dengue virus in human and mosquito tissues is critical to fully understand virus-host relationships, disease severity, viral fitness, and pathogenesis. In recent years, RNA copy number measured by quantitative real time-polymerase chain reaction has been used to measure dengue virus concentration in vitro and in vivo. In this study, we detail important differences in the measurement of viral growth kinetics in Vero and C6/36 tissue cultures, in Aedes aegypti mosquitoes, and in viremic human sera using RNA genomic equivalents and mosquito infectious dose 50 (MID50). Although there was reasonably good correlation between the two methods, RNA copy number was 2 to 5 logs greater than infectious virus titers. These differences varied significantly depending on virus strain, viral platform, infectious virus assay, and viral growth phase. The results have important implications for the correct interpretation of biological and epidemiological data from experimental and clinical studies, and show that genomic equivalents should be interpreted with caution when used as a proxy for infectious virus in such studies.