Strongly inwardly rectifying potassium channels are blocked by intracellular polyamines with a uniquely steep voltage dependence. An understanding of the fundamental details underlying the voltage dependence of polyamine block requires a constrained structural description of the polyamine-binding site. With this goal in mind, we previously used a "blocker protection" approach to examine the effects of polyamine occupancy on the rate of MTSEA modification of cysteine residues located at pore-lining sites in a strongly rectifying Kir channel (Kir6.2[N160D]). In the study presented here, we focused this strategy to characterize the effects of polyamine analogs that are similar in size to spermine on the rate of MTSEA modification. The observed protection profile of spermine is identical to that previously reported, with spermine occupancy inhibiting MTSEA modification of residue 157C, which is deep in the Kir pore, but having little effect on modification rates of 164C or 169C, closer to the intracellular side of the inner cavity. Remarkably, slightly longer synthetic spermine analogs (BE-spermine, CGC-11098) significantly increased the protection observed at position 164C. The extended protection profile observed with slightly extended polyamine analogs significantly enhances the resolution of our previous mapping efforts using the blocker protection approach, by eliminating uncertainties regarding the blocked conformations of the much longer polyamines that were used in earlier studies. For all short polyamine analogs examined, modification at the entrance to the inner cavity (169C) was unaffected by blocker occupancy, although blocker dissociation was dramatically slowed by partial modification of this site. These data support the validity of a blocker protection approach for mapping polyamine-binding sites in a Kir pore, and confirm that spermine binds stably at a deep site in the inner cavity of strongly rectifying Kir channels.