EthR is a mycobacterial repressor that limits the bioactivation of ethionamide, a commonly used anti-tuberculosis second-line drug. Several efforts have been deployed to identify EthR inhibitors abolishing the DNA-binding activity of the repressor. This led to the demonstration that stimulating the bioactivation of ETH through EthR inhibition could be an alternative way to fight Mycobacterium tuberculosis. We propose a new SPR methodology to study the affinity between inhibitors and EthR. Interestingly, the binding between inhibitors and immobilized EthR produced a dose dependent negative SPR signal. We demonstrated that this signal reveals the affinity of the small molecules for the repressor. The affinity constants (KD) correlated with their capacity to inhibit the binding of EthR to DNA. We hypothesize that conformational changes of EthR during ligand interaction could be responsible for this SPR signal. Practically, this unconventional result open perspectives to the development of SPR assay that would at the same time tough on the structural changes of the target upon binding with an inhibitor and on the binding constant of this interaction.