Here, we report the validation of a new reporter cell line, Hec1a-IFNB-Luc, for use in inhibition studies of various flaviviruses relevant to human pathology. The reporter system allows the detection of viral replication after luciferase gene activation driven by an interferon beta (IFN-β) promoter. We found the reporter cell line to be highly responsive to all 10 flaviviruses tested, including the 4 dengue virus serotypes. The applicability of the Hec1a-IFNB-Luc reporter cell line for serodiagnostic purposes in neutralizing antibody assays was confirmed by comparison of its sensitivity and specificity to those of "gold-standard," clinically applied, cytopathic effect-based assays, showing comparable performances. The reporter cell line used for the assessment of viral inhibition by small-molecule antiviral compounds was also confirmed, and the sensitivity of the Hec1a-IFNB-Luc reporter cell line was compared to those from published data reporting on the activity of the antivirals in various other assays, indicating that the Hec1a-IFNB-Luc reporter cell line allowed the determination of the inhibitory capacity at least as sensitive as alternative assays. By measuring luciferase activity as a proxy for viral replication, the reporter cell line allows early detection, reducing the time to results from often 5 to 7 days to 3 days, without the need for optical inspection of cytopathic effects, which often differ between viruses and cell lines, streamlining the development of flavivirus assays. IMPORTANCE The Hec1a-IFNB-Luc reporter cell line allows the detection of all 10 flaviviruses tested, including the 4 dengue virus serotypes. Its use for serodiagnostic purposes, measuring neutralizing antibody activity in sera, and the assessment of the antiviral activities of small-molecule compounds was confirmed, and it was found to be comparable to clinically applied assays. The Hec1a-IFNB-Luc reporter cell line allows the rapid and quantitative determination of antiviral effects on multiple human pathological flaviviruses using a single protocol.