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Simple Summary In stress and disease situations, cells must rapidly and in a coordinated manner change their gene expression patterns to respond adequately. The NF-κB system comprises five transcription factors that are released from the cytosol to enter the nucleus in response to a wide range of extracellular stimuli via a complex cytosolic signaling system. In the nucleus, activated NF-κB dimers bind to specific chromatin loci across the entire genome and induce the expression of a broad repertoire of genes that regulate immune and inflammatory responses. Consistent with its biological importance, the extent of NF-κB activity is regulated and controlled at multiple levels. The aim of this review is to comprehensively present and discuss the currently available conceptual and methodological approaches to monitor the molecular activation status of the NF-κB system, including multi-level single cell analysis. Abstract The NF-κB signaling system plays an important regulatory role in the control of many biological processes. The activities of NF-κB signaling networks and the expression of their target genes are frequently elevated in pathophysiological situations including inflammation, infection, and cancer. In these conditions, the outcome of NF-κB activity can vary according to (i) differential activation states, (ii) the pattern of genomic recruitment of the NF-κB subunits, and (iii) cellular heterogeneity. Additionally, the cytosolic NF-κB activation steps leading to the liberation of DNA-binding dimers need to be distinguished from the less understood nuclear pathways that are ultimately responsible for NF-κB target gene specificity. This raises the need to more precisely determine the NF-κB activation status not only for the purpose of basic research, but also in (future) clinical applications. Here we review a compendium of different methods that have been developed to assess the NF-κB activation status in vitro and in vivo. We also discuss recent advances that allow the assessment of several NF-κB features simultaneously at the single cell level.