TRPM8 has been implicated in nociception and pain and is currently regarded as an attractive target for the pharmacological treatment of neuropathic pain syndromes. A series of analogues of N, N'-dibenzyl tryptamine 1, a potent TRPM8 antagonist, was prepared and screened using a fluorescence-based in vitro assay based on menthol-evoked calcium influx in TRPM8 stably transfected HEK293 cells. The tryptophan derivative 14 was identified as a potent (IC50 0.2 ± 0.2 nM) and selective TRPM8 antagonist. In vivo, 14 showed significant target coverage in both an icilin-induced WDS (at 1-30 mg/kg s.c.) and oxaliplatin-induced cold allodynia (at 0.1-1 μg s.c.) mice models. Molecular modeling studies identified the putative binding mode of these antagonists, suggesting that they could influence an interaction network between the S1-4 transmembrane segments and the TRP domains of the channel subunits. The tryptophan moiety provides a new pharmacophoric scaffold for the design of highly potent modulators of TRPM8-mediated pain.