Migraine is a highly disabling pain disorder with huge socioeconomic and personal costs. It is genetically heterogenous leading to variability in response to current treatments and frequent lack of response. Thus, new treatment strategies are needed. A combination of preclinical and clinical data indicate that ATP-sensitive potassium (K) channel inhibitors could be novel and highly effective drugs in the treatment of migraine. The subtype Kir6.1/SUR2B is of particular interest and inhibitors specific for this cranio-vascular K channel subtype may qualify as future migraine drugs. Historically, different technologies and methods have been undertaken to characterize K channel modulators and, therefore, a head-to-head comparison of potency and selectivity between the different K subtypes is difficult to assess. Here, we characterize available K channel activators and inhibitors in fluorescence-based thallium-flux assays using HEK293 cells stably expressing human Kir6.1/SUR2B, Kir6.2/SUR1, and Kir6.2/SUR2A K channels. Among the openers tested, levcromakalim, Y-26763, pinacidil, P-1075, ZM226600, ZD0947, and A-278637 showed preference for the K channel subtype Kir6.1/SUR2B, whereas BMS-191095, NN414, and VU0071306 demonstrated preferred activation of the Kir6.2/SUR1 subtype. In the group of K channel blockers, only Rosiglitazone and PNU-37783A showed selective inhibition of the Kir6.1/SUR2B subtype. PNU-37783A was stopped in clinical development and Rosiglitazone has a low potency for the vascular K channel subtype. Therefore, development of novel selective K channel blockers, having a benign side effect profile, are needed to clinically prove inhibition of Kir6.1/SUR2B as an effective migraine treatment.