Understanding the dynamics of micro- and nanometer-sized objects like molecules, particles, and living cells in biological systems and biomaterials has become a key component in biomedical research. Consequently, significant progress has been made for the development of imaging platforms, fluorescent probes, and computational tools to visualize and quantify biological processes at different length and time scales. However, despite such advances, achieving a reliable measurement accuracy on the dynamic behavior of these microscopic vehicles in diverse biological contexts is challenging. Subsequently, the motivation behind this dissertation is to develop new robust microfluidic velocimetry techniques to investigate molecular transport and cell migration within an in-vitro microfluidic platform.