Anchorage-independent cultures provide insights into cell proliferation, differentiation, and tumorigenesis beyond traditional two-dimensional models by mimicking parts of the extracellular matrix (ECM). The soft agar colony formation assay enables cells to proliferate in a three-dimensional manner resulting in metabolic phenotypes that are distinct from traditional monolayer cultures. Here, we established a soft agar colony formation assay with subsequent cell isolation to analyze mitochondrial metabolism, metabolic fluxes, morphology, and gene expression within the same sample. We applied mass spectrometry and tracing approaches to decipher carbon utilization for tricarboxylic acid (TCA) cycle metabolism. We also quantified the alteration of immune-related genes in response to inflammatory stimuli in soft agar cultures that might be relevant to autoimmune diseases, which are frequently associated with inflammatory environments and may contribute insights into chronic inflammation and immune cell survival that parallel tumorigenic processes. Our methodology offers a robust model to better understand cell metabolism and function of anchorage-independent cultures that may contribute to the development of new treatment strategies.