Cytokinin (CK) hormones are one of the main plant morphogenetic signals. CK signalling involves a phospho-transfer, triggered by the binding to histidine kinase receptors. The phosphate is transferred to histidine phosphotransferase proteins (AHP), and then from AHPs to response regulators (RRs), transcription factors that will activate target genes. This pathway is non-linear as it is regulated by multiple negative feedbacks. The current knowledge about this pathway results mostly from qualitative analysis of loss-of-function mutants, and of transient expression assays in isolated plant cells (protoplasts), or in homologous systems, such as bacteria and yeast. Here, we designed and built a platform free from the multiple regulatory interactions with other pathways occurring in planta, by reconstructing the full Arabidopsis thaliana CK signalling pathway in mammalian cells. This platform allowed us to quantitatively characterize functional CK signalling regulators. We notably investigated the receptor selectivity to different CKs but also explored the mode of action of three RRs acting as repressors, and of AHPs, allowing to identify novel roles for these proteins. We also assayed quantitatively the interaction between signalling effectors, allowing us to explore the role of a conserved RR residues in the interaction, and the effect of the formation of different complex on the CK pathway activity. Our results were further validated in protoplasts, demonstrating the predictive power of our synthetic biology platform for exploration of plant signalling pathways.