One important obstacle in using softwood in biorefineries is its recalcitrance to enzymatic saccharification. In the present work, Thermothielavioides terrestris LPH172 was cultivated on three different steam-pretreated spruce materials and on non-treated spruce, harboring different hemicellulose content and structural characteristics. The aim of the work was to map the substrate-induced changes in the secretome of T. terrestris when grown on differently treated spruce materials and evaluate the hydrolytic efficiency of the secretome as supplement of a commercial enzyme mixture. The cultivation of T. terrestris was monitored by enzyme activity measurements of endo-cellulase, endo-xylanase, endo-mannanase, laccase and peroxidase. Proteomic analysis was performed on the secretomes induced by these materials to map the differences in enzyme expression.