Palaeohistology has traditionally relied on the qualitative assessment of fossil bone microarchitecture, limiting the resolution and reproducibility of anatomical and physiological analyses. In this dissertation, I apply quantitative polarized light microscopy (qPLM) —using a liquid-crystal variable retarder-enabled system— to the fossil record for the first time. This technique enables a detailed analysis of collagen fibre orientation and distribution in fossil bone. Using qPLM analysis, I identified a non-linear relationship between birefringent properties and the process of permineralization. This transition is marked by a shift from collagen as the primary source of birefringence in modern specimens to hydroxyapatite in permineralized material. Furthermore, qPLM was shown to effectively identify anatomical features such as entheses, lines of arrested growth, and pathology. The orientation and distribution of collagen fibres in these structures provided key physiological insights, including the direction of muscular effort. By quantifying collagen fibre orientation at entheses, qPLM offers a means of informing biomechanical analysis from an isolated skeletal element, such as the moment arm calculations performed here. An extensive review of the historical use of Witmer’s extant phylogenetic bracket approach highlighted a misuse rate exceeding 50%. By directly quantifying key physiological aspects of fossil vertebrate anatomy, qPLM reduces reliance on inferential soft tissue reconstructions, addressing critical methodological gaps and improving the accuracy of paleobiological interpretations. This research demonstrates the transformative potential of qPLM in palaeohistology, providing a reproducible, high-resolution approach to studying fossil bone microarchitecture and its functional implications.