The Biotin Identification (BioID) method is an in vivo labeling strategy for the identification of cellular microenvironments in animal cells, plants, yeast and filamentous fungi. With this method, the protein of interest (POI) is fused to a promiscuous biotin ligase. The ligase biotinylates proximal proteins in a cumulative manner, which are then captured from cell lysates by affinity-binding and identified by mass spectrometry. This allows the identification of POI co-localizing proteins, including weakly or transiently binding ones (Roux et al., 2012; Branon et al., 2018). In our recent research, we performed BioID experiments with the TurboID biotin ligase expressed in the filamentous fungus Sordaria macrospora (Hollstein et al., 2022). Here, we tested a smaller variant of TurboID biotin ligase called miniTurboID for its application S. macrospora. For a proof of principle, we used the well-characterized striatin interacting phosphatase and kinase (STRIPAK) complex as molecular microenvironment. In detail, the STRIPAK complex interactor 1 (SCI1) was fused to the codon-optimized TurboID or the miniTurboID biotin ligase. These fusion proteins complemented a sterile Δsci1 deletion strain to restore fertility. A BioID experiment with LC-MS analysis showed significant enrichment of the SmSTRIPAK components PRO11, SmMOB3 and PRO22 in all biological replicates of SCI1-TurboID and SCI1-miniTurboID, while being absent in the free TurboID control. This demonstrates that both, TurboID and the smaller variant miniTurboID, can be applied in the filamentous ascomycete S. macrospora. We provide a step by step protocol for the preparation of BioID samples for LC-MS analysis in the MethodsX article.