After more than 40 years since the signing of the National Cancer Act in 1970, cancer remains a formidable challenge. Cancer is currently the second most common cause of death in the United States, and worldwide cancer cases are projected to rise 50% between 2012 and 2030 [1-2]. While researchers have dramatically expanded our understanding of the biology of cancer, they have also revealed the staggering complexity and difficulty of developing successful treatments for the disease. More complex assays involving three dimensional cell culture offer the potential to model complex interactions, such as those involving the extracellular matrix (ECM), chemical concentration gradients, and the impact of vascularization of a tissue mass. Modern cancer assays thus promise to be both more accurate and more complex than previous models. One promising newly developed type of assay is microfluidics. Microfluidic devices consist of a silicone polymer stamp bonded to a glass slide. The stamp is patterned to produce a network of channels for cell culture. These devices allow manipulation of liquids on a sub-millimeter level, allowing researchers to produce a tightly controlled 3D microenvironment for cell culture. Our lab previously developed a microfluidic device to measure cancer cell invasion in response to a variety of signals and conditions. The small volume associated with microfluidics offers a number of advantages, but simultaneously make it impractical to use certain traditional cell analysis procedures, such as Western Blotting. As a result, measuring protein expression of cells in the microfluidic device was a continuing challenge. In order to expand the utility of microfluidic devices, it was therefore very enticing to develop a means of measuring protein expression inside the device. One possible solution was identified in the technique of In-Cell-Western blotting (ICW). ICW consists of using infrared-fluorescently stained antibodies to stain a protein of interest. This signal is measured using an infrared laser scanner, producing images that can be analyzed to quantitatively measure protein expression. ICW has been well validated in traditional 2D plate culture conditions, but has not been applied in conjunction with microfluidic devices. This project worked to evaluate In-Cell-Western blotting for use in microfluidic devices as a method of quantifying protein expression in situ.