SummaryFluorescent labeling of proteins is a widespread approach for the microscopic examination of protein function, expression, and localization in the cell. Here, we present a protocol for the labeling of hemagglutinin (HA)-tagged protein of interest (POI) with the single-chain antibody (scFv) 2E2 fused to different fluorescent proteins (FPs) inSaccharomyces cerevisiae. We describe steps for expressing 2E2-FP, and HA tagging and labeling of POI. We detailin vivofluorescent imaging of proteins at different cellular compartments and with diverse expression levels.For complete details on the use and execution of this protocol, please refer to Tsirkas et al. (2022).1