All organisms contain thioredoxin (TRX), a regulatory thiol:disulfide protein that reduces disulfide bonds in target proteins. Unlike animals and yeast, plants contain numerous TRXs for which no function has been assignedin vivo. Recentin vitroproteomic approaches have opened the way to the identification of >100 TRX putative targets, but of which none of the numerous plant TRXs can be specifically associated. In contrast,in vivomethodologies, including classical yeast two-hybrid (Y2H) systems, failed to reveal the expected high number of TRX targets. Here, we developed a yeast strain named CY306 designed to identify TRX targetsin vivoby a Y2H approach. CY306 contains a GAL4 reporter system but also carries deletions of endogenous genes encoding cytosolic TRXs (TRX1andTRX2) that presumably compete with TRXs introduced as bait. We demonstrate here that, in the CY306 strain, yeast TRX1 and TRX2, as well asArabidopsisTRX introduced as bait, interact with known TRX targets or putative partners such as yeast peroxiredoxins AHP1 and TSA1, whereas the same interactions cannot be detected in classical Y2H strains. Thanks to CY306, we also show that TRXs interact with the phosphoadenosine-5-phosphosulfate (PAPS) reductase MET16 through a conserved cysteine. Moreover, interactions visualized in CY306 are highly specific depending on the TRX and targets tested. CY306 constitutes a relevant genetic system to explore the TRX interactomein vivoand with high specificity, and opens new perspectives in the search for new TRX-interacting proteins by Y2H library screening in organisms with multiple TRXs.