Mushrooms represent a promising source of bioactive macromolecules with numerous biological properties, including prebiotic potential. Although most studies focus on polysaccharide extracts, emerging evidence suggests that diverse mushroom macromolecules, such as proteins, peptides, fatty acids, and polyphenolic compounds, may also exert prebiotic effects. This chapter comprehensively explores the methodological approaches for the evaluation of the prebiotic properties of edible fungi, emphasizing the gastrointestinal simulation and gut microbiota modulation approaches. The standardized INFOGEST protocol simulates human gastrointestinal conditions, helping to understand the bioaccessibility of mushroom-derived compounds. Moreover, in vitro intestinal permeability assays are used to determine the colon-available fraction. Human fecal fermentation assays are frequently employed to assess mushrooms’ prebiotic potential and its impact on gut microbiota modulation. These studies typically monitor key parameters, including their impact on bacterial population dynamics, production of SCFAs, and other relevant metabolic variations (e.g., amino acid metabolism). By integrating in vitro digestion and fermentation models with advanced molecular biology, microbiological, and chemical techniques, this chapter provides a systematic outline for evaluating the prebiotic potential of mushroom compounds and understanding their interaction with gut microbiota composition and function.