Background: Over the last decade, per- and polyfluoroalkyl substances (PFASs) have become one of the most heavily investigated persistent organohalogen compound class of environmental concern. However, knowledge about their toxicology is still scarce, although PFASs as individual compounds and their industrial mixtures were shown to exert effects on the thyroid hormone system. Methods: In vitro toxicity potency factors were established for thyroid hormone transport disruption potential using the novel TTR-TRβ CALUX® bioassay for major PFASs. We assessed technical PFASs mixtures, including aqueous film-forming foam (AFFF) surfactants and chromium mist suppressants (CMS) applications with and without total oxidizable precursor (TOP) by TTR-TRβ CALUX® assay for their thyroid hormone transport disrupting potential. Results: All PFASs listed in the German guideline for drinking water (German Environment Agency, 2017) affected the T4 binding to TTR, an important plasma thyroid hormone transport protein. For all tested PFASs, potency factors based on PC80 values relative to PFOA could be obtained and ranged between PFBA (0.0018) and PFOS (2.0). Applying in vitro potency factors obtained from the present in vitro TTR-TRβ CALUX® assay study and recently reported in vivo potency factors (Zeilmaker et al., 2018; Bil et al., 2021) on the above-mentioned German guideline for PFAS in drinking water, showed that the cumulative effect-based trigger values (in vivo and in vitro) are comparable (3.0 vs. 2.9 to 4.6 μg PFOA-EQ/l). Additionally, AFFF surfactants and CMS with and without TOP assay were tested. Highest activities were found in the older AFFF surfactants (2013/2014) due to higher PFOS/PFOA levels, which were already substituted with 6:2 FTS in 2019, resulting in much lower PFOA-EQ levels. As expected also the PFOA-EQ levels increased in the samples with TOP treatment compared to the original AFFF surfactants and CMS as confirmed here by biological and chemical PFOA-equivalents (PFOA-EQ) analysis. Additionally, CMS (which have been used in the electroplating chromium industry since the 1950s) as well as PFOS-free, but not PFAS-free fume suppressants (such as Fumetrol® 21) have been tested in the TTR-TRβ CALUX® assay and showed much lower activity levels then the AFFFs, confirmed by the similar potency determination based on chemical PFASs analysis followed by transformation to PFOA-EQ for comparison. The potency factor of 6:2 FTS, which is the main substitute for PFOS in CMS, indicates that it is approximately 100-times less potent as a thyroid hormone disruptor as compared to PFOS. Conclusion: Potency factors based on PC80 values from TTR-TRβ CALUX® relative to PFOA have been developed for major PFASs. In AFFF surfactants and CMS a trend of higher activities with higher amounts of PFOS and PFOA have been found. PFOA and PFOS showed high responses in the TTR-TRβ CALUX® assay and had the largest contributions to the PFOA-EQs in the AFFF surfactants and CMS applications. Using potency factors as determined in the TTR-TRβ CALUX® to convert PFASs assessed by chemical analysis to PFOA-EQ led to comparable results as compared to the results from PFASs measured directly by the TTR-TRβ CALUX® assay. This study supports the claim that semiquantitative effect- and group-based in vitro CALUX bioanalysis tools can be applied effectively to assess industrial products containing complex mixtures with PFAS compounds for which no instrumental analysis are established, and for many compounds where in vitro toxicity data are not yet available.