The composition of the striatin-interacting phosphatases and kinases (STRIPAK) signaling complex is highly similar in all eukaryotes and controls for the phosphorylation and dephosphorylation of target proteins. Associated with STRIPAK are germinal center kinases (GCK), some of which are part of conserved downstream signaling pathways. Using protein samples from wild type and three STRIPAK mutants, we applied absolute quantification by parallel reaction monitoring (PRM) to analyse phosphorylation site occupancies.