Quantifying mechanically-induced changes in the tricuspid valve extracellular matrix (ECM) structural components, e.g. collagen fiber spread and distribution, is important as it determines the overall macro-scale tissue responses and subsequently its function/malfunction in physiological/pathophysiological states. For example, functional tricuspid regurgitation, a common tricuspid valve disorder, could be caused by elevated right ventricular pressure due to pulmonary hypertension. In such patients, the geometry and the normal function of valve leaflets alter due to chronic pressure overload, which could cause remodeling responses in the ECM and change its structural components. To understand such a relation, we developed an experimental setup and measured alteration of leaflet microstructure in response to pressure increase in porcine tricuspid valves using the small angle light scattering technique. The anisotropy index, a measure of the fiber spread and distribution, was obtained and averaged for each region of the anterior, posterior, and septal leaflet using four averaging methods. The average anisotropy indices (mean ± standard error) in the belly region of the anterior, posterior, and septal leaflets of non-pressurized valves were found to be 12 ± 2%, 21 ± 3% and 12 ± 1%, respectively. For the pressurized valve, the average values of the anisotropy index in the belly region of the anterior, posterior, and septal leaflets were 56 ± 5%, 39 ± 7% and 32 ± 5%, respectively. Overall, the average anisotropy index was found to be higher for all leaflets in the pressurized valves as compared to the non-pressurized valves, indicating that the ECM fibers became more aligned in response to an increased ventricular pressure. Mechanics plays a critical role in development, regeneration, and remodeling of tissues. In the current study, we have conducted experiments to examine how increasing the ventricular pressure leads to realignment of protein fibers comprising the extracellular matrix (ECM) of the tricuspid valve leaflets. Like many other tissues, in cardiac valves, cell-matrix interactions and gene expressions are heavily influenced by changes in the mechanical microenvironment at the ECM/cellular level. We believe that our study will help us better understand how abnormal increases in the right ventricular pressure (due to pulmonary hypertension) could change the structural architecture of tricuspid valve leaflets and subsequently the mechanical microenvironment at the ECM/cellular level.