The aim of this work was to compare the biohydrogen production potential of undetoxified and detoxified acid hydrolysates from A. tequilana bagasse. Detoxification was carried out with activated carbon at different concentrations and pH values. Results indicated that pH was not a significant variable, while the lowest evaluated concentration of activated carbon (1% p/v) significantly promoted the highest removal of acetic acid (89%) with minimal losses of fermentable sugars. Regarding dark fermentation experiments, central composite designs were used to optimize COD and pH variables for both substrates, undetoxified and detoxified hydrolysates (activated carbon 1% p/v and pH 0.6). At optimal conditions, the detoxified hydrolysate produced 33% more biohydrogen than the undetoxified one. Hydrogen molar yields were 1.71 and 1.23 mol H₂/mol_sugar, respectively. This improvement was correlated to changes in metabolic byproducts, since the detoxified hydrolysate produced only acetic and butyric acids, while lactic acid was detected in the undetoxified hydrolysate.