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_ATP) 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_ATP channel subtype may qualify as future migraine drugs. Historically, different technologies and methods have been undertaken to characterize K_ATP channel modulators and, therefore, a head-to-head comparison of potency and selectivity between the different K_ATP subtypes is difficult to assess. Here, we characterize available K_ATP 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_ATP channels. Among the openers tested, levcromakalim, Y-26763, pinacidil, P-1075, ZM226600, ZD0947, and A-278637 showed preference for the K_ATP 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_ATP 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_ATP channel subtype. Therefore, development of novel selective K_ATP channel blockers, having a benign side effect profile, are needed to clinically prove inhibition of Kir6.1/SUR2B as an effective migraine treatment.