The PKC1-Cell Wall Integrity Pathway (PKC1-CWIP) is essential for survival and maintenance of cell wall integrity in pathogen and model yeast cells. In S. cerevisiae, the main protein sensors of the PKC1-CWIP, Wsc1 and Mid2, are responsible for the activation of the pathway in the presence of cell wall stress. Therefore, Wsc1p and Mid2p are an attractive target for antifungal drugs because they contribute to cell viability. The purpose of this research is to validate the sensor-interacting proteins previously identified by integrated Membrane Yeast Two-Hybrid (iMYTH) using Tandem Affinity Purification coupled to Mass Spectrometry (TAP-MS). To validate the iMYTH interactions, Wsc1-TAP and Mid2-TAP bait strains were co-precipitated by affinity to calmodulin sepharose beads and compared with a wild-type BY4741 strain under normal growth conditions. Results showed that Wsc1p and Mid2p baits were recovered. One protein, encoded by RPL40A gene was confirmed as a Mid2p interactor. Twenty new interactors were identified for Wsc1p and 23 new interactors for Mid2p in the TAP-MS. Ndr1p was the only interactor found to be previously annotated in the BioGRID. In conclusion, the Mid2-Rpl40a interaction was validated by both iMYTH and TAP-MS. The novel Wsc1p and Mid2p-interacting proteins identified come from a range of different biological processes, including stress response, protein synthesis and cell wall organization.This research was supported by University of Puerto Rico-Medical Sciences Campus, University of Toronto, University of Regina and NIH grants G12MD007600, U54MD007587, P20GM103475 and R25GM061838.