The reprogramming of cell metabolism is a hallmark of cancer. NF-κB transcription factors coordinate the host defense responses to stress, injury, and infection. They also play a central role in oncogenesis, at least in part by regulating cell metabolism and the adaptation to energy stress conditions in various types of cancer, such as colorectal carcinoma (CRC). Here, we describe the XF Cell Mito Stress Test methodology aimed at characterizing the metabolic and bioenergetic profile of CRC cells following the silencing of the essential NF-κB subunit, RelA. This methodology may also be applied to other cancers to reveal novel core vulnerabilities of malignant cells.