Hairy root transformation represents a valuable tissue culture system for plant biotechnology in species that are difficult to propagate via standard seed-based methods. Infection by an Agrobacterium strain carrying a Root-inducing (Ri) plasmid and a subsequent transfer of T-DNA into the plant genome induces the formation of hairy roots at the wounding site of the host plant. Hairy roots can be used to directly analyze a transgene of interest or processed for generation of transgenic plants. Here, we developed and evaluated a genetic reporter system for investigation of auxin and cytokinin in rapeseed (Brassica napus) tissues. While these major phytohormones have been widely studied in Arabidopsis thaliana, tools for monitoring their spatial signaling activity in crop species remain limited. We constructed two types of GUS-based auxin-responsive reporters driven either by a synthetic pDR5cc or a composite pBnIAA promoters. Each reporter was tested with or without the presence of a 5’UTR. Reporter activity was induced applying exogenous auxin, and GUS expression in hairy roots was visualized via histochemical staining. To insert a new trait, hairy root system can be utilized to re-transform regenerated transgenic plants. To monitor signaling of both auxin and cytokinin in the hairy root tips, we re-transformed the B.napus regenerant carrying the TCSv2:3×VENUS cytokinin reporter with Agrobacterium strain containing a binary vector encoding the DR5-Tag-BFP auxin reporter. Phytohormone sensitivity of both reporters was assessed by measuring the fluorescence intensity upon exogenous application of auxin and cytokinin.