In this assay study design, activation through stimulation or inhibition of the Nuclear Factor κB (NF-κB) pathway demonstrates translocation of NF-κB protein from the cytoplasm to nucleus as measured using automated fluorescent microscopy computer-assisted image analysis technology better known as high content screening (HCS), High Content Analysis (HCS), High Content Imaging (HCI), or Image Cytometry (IC). This approach offers a better understanding of novel drug targets by examining the sub-cellular spatial distribution of the target proteins and the effects of target perturbation on cellular processes. Cell imaging provides multi-probe detection and is advantageous over other methods in quantifying spatial measurements such as protein translocation or redistribution, receptor internalization, cell morphology, cell motility, cell cycle, cell signaling, and others. The limitation of traditional microscopy and image analysis has been a log jam of sample throughput, day-to-day variability, poor standardization and of the need for specialized personnel to operate and oversee instrumentation. Automated fluorescent microscopy systems offer one solution for studying cell function in a “high content screening” (HCS) mode that allows the capability of measuring multiple cellular characteristics in a non-biased fashion. This chapter details the assay development, optimization, and validation for running a compound screening campaign and includes descriptions of the cell model, stimuli to activate NF-κB pathway, time course kinetics, effects of serum on assay window, validation of proinflammaory cytokines, reference control inhibitor compound optimization, and examples of nomenclatures used by many manufactures of HCS instruments or software algorithms.