The identification and quantification of specific proteins in complex samples are critical processes in discovering, understanding, and ultimately addressing, the underlying nature of human health and disease. The western blot is a fundamental method used for these processes due to the sensitivity and specificity enabled by combining protein sizing with immuno-detection. Despite being widely- used, the western blot has limits related to required sample size, throughput, information content, quantification, and repeatability, which hinder many research efforts. An example is the challenge in determining expression levels of multiple proteins in the very small samples associated with pancreatic islets and mouse biopsies used in research related to diabetes and obesity. Next Generation Western (NGW) is an innovative approach to alleviate limitations of traditional western blotting using a novel method that automates and miniaturizes the process of electrophoresis followed by direct-blotting onto a moving capture medium for subsequent immuno-probing and detection. To demonstrate feasibility of the NGW technology, the specific aims are to (1) enable separation resolution comparable to current western blot performance, (2) develop a sample injection/loading scheme to optimize limit-of-detection for a wide range of protein sizes, and (3) demonstrate the power of the technology by analyzing the total and phosphorylated expression levels of five proteins in a single cell lysate using a sample size ~50-fold smaller than required by current technology. LI-COR Biosciences is well established in the western blot market with worldwide sales and support of instrumentation and reagents. After demonstrating feasibility, a product based on this technology will be developed in collaboration with the Kennedy lab to enable western blots with significantly less sample (nanograms instead of micrograms), reduced complication (automated separation/transfer), and improved throughput and content (48+ lanes per current western blot lane).