Perfluorinated alkyl acids (PFAAs) are used in many household products including food contact materials. Hence, humans are continuously exposed, and the PFAAs are accumulated in human serum with half-lives up to 8.8 years. In humans, high PFAA serum levels have been associated with an increased risk of breast cancer and other adverse health effects such as lower birth weight and longer time to pregnancy which might be related to disruptions of various hormonal systems. For instance, direct cell exposure studies in vitro suggest that some PFAAs can transactivate the estrogen receptor (ER), antagonize the androgen receptor (AR) and has the potential to interfere with Thyroid Hormone and Aryl-hydrocarbon Receptor functions. Moreover, the PFAAs also showed cellular oxidative stress potential. Humans are exposed to an array of PFAAs, and the quantity and combination of these PFAAs in human serum differs between individuals. Hence, the toxicological studies of single PFAAs and simple mixtures might be insufficient to predict how the actual mixtures of PFAAs may affect humans. To get a better evaluation of the actual mixture effects, we developed a method to extract the actual mixture of PFAAs from human serum. Preliminary results showed that 17% of the PFAA serum fractions from pregnant women could significantly transactivate the ER, and 94% of the fractions could further increase the transactivity induced by the potent ER ligand 25 pM 17β-estradiol. As part of the international FETOTOX project (http://fetotox.au.dk/), we are currently extracting the actual PFAA serum mixture from 700 pregnant women to further elucidate the potential of the serum PFAA mixture to transactivate the ER at the levels found in human serum. We suggest that our method can in the future be used to study the effects of the actual serum PFAA mixture on both steroid hormone actions as well as other hormonal systems e.g. thyroid hormone function. In the current review we will discuss how our recently developed PFAA extraction method might be used in future research to assess the endocrine impact of PFAAs on human health.