A crucial step in quantitative proteomics is an artefact free and reproducible sample preparation protocol, which has to be adapted and optimized to nearly all types of cells. Here we provide a sample preparation method for quantitative proteomics of cellular fungi. Two different protein extraction methods were compared with focus on reproducibility, minimized proteolytic degradation and protein losses during the sample preparation. In the first preparation the cells were lysed by sonication followed by protein solubilization in “standard” lysis buffer. The second preparation was performed with a SDS-presolubilization step followed by sonication and further boiling, before diluting the sample with lysis buffer. We have shown that the sample preparation for cellular fungi is performed with maximum protein solubilization, higher reproducibility and a reduced proteolytic activity by including a SDS-presolubilization step in the sample preparation protocol.