Many species of the fungal genus Trichoderma are potent mycoparasites, which turns them into successful bio-fungicides for the biological control of plant diseases. Investigations on the underlying signal transduction pathways revealed that G protein signalling and pathways involving mitogen-activated protein kinases (MAPKs) play important roles in the recognition of the host fungus and activation of the mycoparasitic response. Mutants missing the MAPK-encoding tmk1 gene show only reduced mycoparasitic abilities. To gain detailed knowledge on the phosphoproteome of Trichoderma atroviride, a workflow was established to identify phosphoproteins in different fungal samples (wild-type with induced and un-induced mycoparasitic activity, -tmk1 mutant with reduced mycoparasitism) which may contribute to mycoparasitism. After careful investigation of different sample preparation methods like TiO2 and IMAC enrichment of phosphorylated peptides and proper measurement conditions for mass spectrometric analysis, the phosphoproteome of Trichoderma atroviride was analyzed by means of matrix-assisted laser desorption/ionization - time of flight - mass spectrometry and the use of post source decay fragmentation of peptides to identify protein species. With this approach it was possible to identify phosphoproteins in all samples of interest and conclusions on their contribution to signaling events during the activation of the mycoparasitic cascade were drawn. The developed methodology allowed to gather a first overview of proteins, which are likely to be regulated by Tmk1 MAPK. Thus it can be stated, that there is a potential to gain further information on the phosphoproteome by following the proposed workflow which can subsequently lead to a better understanding of underlying mechanisms involved in mycoparasitism.