Protein–protein interactions play critical roles in biological processes. We previously developed the Pil1 co-tethering assay, an imaging-based method to detect protein–protein interactions in living Schizosaccharomyces pombe cells. This assay leverages the distinct localization pattern of the Pil1 protein by fusing a bait protein to Pil1 and examining whether a prey protein co-localize with the Pil1-fused bait. Here, we present an improved protocol of the Pil1 co-tethering assay. In this protocol, modified stable integration vectors (SIVs) with a NotI site as the linearization site are used to express bait and prey proteins. We expect that this protocol will enhance the application of the Pil1 co-tethering assay for studying protein–protein interactions.