Epidemiological evidence supports associations between prenatal exposure to environmental organic chemicals and childhood health impairments. Unlike the common choice of biological matrices such as urine and blood that can be limited by short half-lives for some chemicals, teeth provide a stable repository for chemicals with half-life in the order of decades. Given the potential of the tooth bio-matrix to study long-term exposures to environmental organic chemicals in human biomonitoring programs, it is important to be aware of possible pitfalls and potential opportunities to improve on the current analytical method for tooth organics analysis. We critically review previous results of studies of this topic. The major drawbacks and challenges in currently practiced concepts and analytical methods in utilizing tooth bio-matrix are (i) no consideration of external (from outer surface) or internal contamination (from micro-odontoblast processes), (ii) the misleading assumption that whole ground teeth represent prenatal exposures (latest formed dentine is lipid rich and therefore would absorb and accumulate more organic chemicals), (iii) reverse causality in exposure assessment due to whole ground teeth, and (iv) teeth are a precious bio-matrix and grinding them raises ethical concerns about appropriate use of a very limited resource in exposure biology and epidemiology studies. These can be overcome by addressing the important limitations and possible improvements with the analytical approach associated at each of the following steps: (i) tooth sample preparation to retain exposure timing, (ii) organics extraction and pre-concentration to detect ultra-trace levels of analytes, (iii) chromatography separation, (iv) mass spectrometric detection to detect multi-class organics simultaneously, and (v) method validation, especially to exclude chance findings. To highlight the proposed improvements we present findings from a pilot study that utilizes tooth matrix biomarkers to obtain trimester-specific exposure information for a range of organic chemicals.