The influenza M2 proton channel is a major drug target, but unfortunately, the acquisition of resistance mutations greatly reduces the functional life-span of a drug in influenza treatment. New M2 inhibitors have been reported that inhibit mutant M2 channels otherwise resistant to the early adamantine-based drugs, but it remains unclear whether and how easy resistance could arise to such inhibitors. We have combined a newly developed proton conduction assay with an established method for selection and screening, both Escherichia coli based, to enable the study of M2 function and inhibition. Combining this platform with two groups of structurally different M2 inhibitors allowed us to isolate drug resistant M2 channels from a mutant library. Two groups of M2 variants emerged from this analysis. A first group appeared almost unaffected by inhibitor: M_089 (N13I, I35L, F47L) and M_272 (G16C, D44H), and the single substitution variants derived from these: I35L, L43P, D44H and L46P. Functionally these resemble the known drug-resistant M2 channels: V27A, S31N and Swine flu. In addition, a second group of tested M2 variants were all still inhibited by drugs but to a lesser extent than wild type M2. MD simulations aided in distinguishing the two groups where drug binding to wild type and the less resistant M2 group showed a stable positioning of the ligand in the canonical binding pose, as opposed to the drug resistant group where the ligand rapidly dissociated from the complex during the simulations.