Flame retardants (FRs), such as polybrominated diphenyl ethers (PBDEs) and phosphate flame retardants (PFRs), are a diverse group of compounds that are used to improve fire safety in many consumer products, such as furniture, textiles, electronics, etc. As these compounds are potentially harmful for human health, there is a need to better understand human exposure. Exposure to environmental contaminants can be monitored by the measurement of external sources of exposures and also by the determination of contaminant levels in human samples. For ethical and practical reasons, noninvasive matrices, such as hair, are preferred but, unfortunately, not widely used due to methodological limitations. A major challenge is sample availability: only small amounts can be sampled per individual. Multi-residue methods are therefore essential in order to determine multiple compounds in low sample amounts. In the framework of the FP7 project (INFLAME), an analytical method for the simultaneous determination of PBDEs and PFRs in human hair has been optimized and validated. Before extraction, hair samples (200 mg) were denaturated in nitric acid (HNO3) for 25 min at 25 °C. Consecutively, the samples were extracted using a mixture of hexane:dichloromethane, and extracts were further fractionated on Florisil. Fraction A which contained PBDEs was additionally cleaned on acidified silica gel and measured by gas chromatography coupled with electron capture negative ionization mass spectrometry (GC-ECNI-MS), while fraction B containing PFRs was directly analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). This approach resulted in recoveries between 81-120% for PBDEs and 75-113% for PFRs (relative standard deviation (RSD) < 16%, n = 9). The optimized multi-residue method has been applied to 20 human hair samples. The obtained results indicated that the levels of PBDEs in hair samples were very low (0.2-12 ng/g) in relation to PBDE levels in human hair samples from other studies and most of the time below the method limit of quantification (LOQm). On the contrary, the PFR levels were relatively high as they were in the range of the levels previously found in dust samples (2-5,032 ng/g hair). We would like to highlight that the contribution of air and dust cannot be neglected (especially in the case of PFRs); therefore, we suggest that hair might be a good indicator of retrospective and integral exposure (which includes atmospheric deposition as well as endogenous mechanisms). Moreover, the aim of our study is focused on exposure assessment and levels detected in hair (independently of whether they come from internal or external exposure) and will significantly contribute to the exposure assessment.