Cancer remains a leading cause of morbidity and mortality worldwide, highlighting the need for alternative therapies with reduced toxicity. Ganoderma lucidum, commonly known as Reishi mushroom, has been recognized for its traditional use in promoting health and longevity and is emerging as a promising medicinal fungus in cancer therapy. The polysaccharides and triterpenoids found in Reishi mushroom have demonstrated potential in inhibiting cancer progression by impairing DNA repair pathways, inhibiting cell proliferation and migration, activating apoptosis, boosting antitumor immunity, and enhancing the susceptibility of cancer cells to conventional therapies. Here, we describe the use of Annexin V assays and Western blot analysis to assess apoptosis. Annexin V staining allows for the distinction between viable, early apoptotic, and late apoptotic/necrotic cells. Furthermore, Western blot analysis is employed to examine the expression of key proteins involved in apoptotic pathways, including caspases and other markers associated with cell survival and death.