Filamentous fungi are widely used in industry as they are key microorganisms in the production of enzymes. Trichoderma reesei is generally considered as the most efficient organism for the production of cellulolytic enzymes to degrade lignocellulosic biomass, in the context of second generation bioethanol production. This work, realised in the framework of a collaboration between IFPEN and PAPPSO, aims at characterizing the extracellular enzyme production of T. reesei, induced by different culture conditions. We used a bottom-up proteomics approach coupling nano liquid chromatography to high resolution mass spectrometry (nano LC-HRMS). X!TandemPipeline, a software developed by PAPPSO was used to perform proteins identification from peptide MS/MS spectra allowing to obtain spectral counting information. Label-free quantification was carried out combining three complementary peptide detection methods: Spectral Counting quantification (SC) obtain by X!TandemPipeline software and, Peak Counting (PC) and eXtracted Ion Chromatogram quantification (XIC). We used MassChroQ software designed for peptide retention time alignment, peak detection and peak integration area. Robust statistical analysis was perfomed by MassChroqR, a dedicated R language package. Secreted proteins from 8 different cultures were analyzed in one injection on a Q-Exactive+ mass spectrometer (Thermo Scientific) totalling 139 proteins identified in all samples. Relative quantification of the different secretomes reveals 113 proteins with significantly variable abundance between cultures and allows to obtain global profiles of the proteins involved in biomass conversion. Relative comparison 2 by 2 helps to better target and understand the difference between conditions.