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This work evaluated the protein quality of shiitake mushroom cultivated on low-nitrogen wood substrates that has previously been shown to enhance biorefinery processes through biological pretreatment. The effects of different initial mushroom substrates (IMSs) on amino acid (AA) content in shiitake fruit bodies (FBs) and spent mushroom substrates (SMSs) were studied using alder, birch and aspen wood substrates with varying nitrogen levels by adding whey (0-2%). Twenty-two AAs, including nine essential AAs (EAAs), were detected and analyzed. Total AA (TAA) content reached up to 13% in FBs and 5% in SMSs. The FBs showed good protein quality according to FAO/WHO guidelines, despite a low nitrogen content (∼0.6%) in IMS. Whey addition increased TAA and EAA levels in FBs while substrate species had distinct effects. Birch generally enhanced AA levels, alder reduced them, and aspen showed AA-specific but mostly positive responses. In SMS, whey addition also increased TAA, but the effects of wood species on TAA generally showed patterns opposite to that observed in FBs. Using 13 IMS chemical constituents as predictive variables in partial least squares regression, five models for AAs in FBs and 15 for SMS were achieved. The C/N ratio and soluble NO2 were major predictors, whereas Klason lignin had the least influence. The results suggested that shiitake SMS is a valuable resource for potential protein extraction and biofuel production.