Recent techniques for detection and quantification of dioxins and dioxin-like compounds involve mainly the costly and time-consuming high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS). To ensure environmental safety, it is extremely crucial to develop high-throughput assays for screening potential contamination of dioxin-like chemicals in various field samples from environments and foods. The Chemical Activated LUciferase eXpression (CALUX) bioassay is an in vitro luciferase-reporter-gene assay for detecting the Toxic Equivalents (TEQ) levels of dioxins and dioxin-like compounds based on their high affinity with aryl hydrocarbon receptor (AhR). As compared with HRGC/HRMS, CALUX bioassay is a fast and low-cost method for high-throughput screening of those highly dioxin-contaminated samples from environment and foods. The technique relies on selection of stable dioxin-responsive cell lines that can maintain their responsiveness to dioxins and dioxin-like compounds. We have established a stable dioxin-responsive cell line, Huh7-DRE-Luc. In the present study, we have determined the capability of this cell line (DRE-luciferase bioassay) in detection of dioxins and dioxin-like compounds by testing with 2,3,7,8-TCDD, CRM powder, EDF-5416, and samples from various matrices (i.e., soil, sediment, and flue gas). Our results showed that the ratio of DRE-Luciferase reading (BIO) to HRGC/HRMS reading (CHE) in testing with 2,3,7,8-TCDD was around 1:1, the ratio of BIO to CHE in testing with CRM powder was around 5:1, and the ratio of BIO to CHE in testing with EDF-5416 was around 3.9:1. Meanwhile, the BIO/CHE ratios in testing with samples from soil, sediment, and flue gas are 31.2, 21.7 and 26.7, respectively. Regarding testing with pure 2,3,7,8-TCDD (with BIO/CHE ratios around 1 and TCDD detection limit of 35.4 pM), Huh7-DRE-Luc is suggested to be a qualified system for detection of dioxins and dioxin-like compounds. However when pre-treatment steps are included in the sample preparation, the BIO/CHE ratios raise markedly. This data suggests that our pre-treatment procedure may interfere with the BIO/CHE ratios. On the basis of these results, the objectives of our future studies are to improve the TCDD detection sensitivity of our DRE-luciferase bioassay system and to find out the factor(s) that may affect the BIO/CHE ratios during the pre-treatment of samples.