Inflammation, a critical adaptive reaction, protects against pathogens, cell dysfunction, and hypersensitivity while facilitating tissue repair. However, persistent infection underpins a spectrum of problems, together with cardiovascular diseases, cancers, diabetes, neurodegeneration, and immune disorders. Traditional anti-inflammatory agents, inclusive of nonsteroidal anti-inflammatory drugs and corticosteroids, even though effective, exhibit significant adverse effects, inclusive of gastrointestinal problems and immunosuppression, prompting the exploration of protective less harmful replacements. Medicinal mushrooms, with their multifaceted bioactive compounds, represent a promising road for managing inflammatory damage. Compounds such as β-glucans, phenolics, terpenoids, and sterols display pronounced anti-inflammatory potential, antioxidant, and immunomodulatory properties. Those mechanisms encompass the inhibition of proinflammatory cytokines (interleukin-6, tumor necrosis factor, and interleukin-1 beta) and modulation of pathways such as NF-κB and MAPK. Moreover, medicinal mushrooms display the capability in neuroprotection, intestine microbiota modulation, and the mitigation of oxidative stress. This chapter elucidates the in vitro evaluation of anti-inflammatory efficacy through the use of the MTT assay, highlighting the defensive properties of mushroom extracts on LPS-triggered anti-inflammatory responses in RAW 264.7 macrophage cells. The defined method offers a strong platform to validate the healing capacity of medicinal mushrooms in the regulation of inflammation.