The ability to evaluate biological processes at multiple levels is crucial to attaining a fundamental understanding of their function in both normal and disease states. It is therefore crucial to acquire reliable, reproducible, and statistically robust data from experimental approaches seeking to gain biological insights. Automated methods of biological analysis offer great promise in this regard, while providing additional benefits such as increased efficiency and reduced intrinsic user bias. This technical seminar will highlight the high-throughput capabilities of the Agilent BioTek Cytation C10 Confocal Imaging Reader to interrogate the SMAD signaling pathways at multiple biological levels. Practical considerations at each level will be discussed, including experimental format and set-up, reagent selection, sample preparation and imaging, image processing, and cellular analysis.