TRPV1 is a polymodal receptor for capsaicin, heat, and other noxious stimuli. Using an iterative approach combining computational docking and functional tests such as thermodynamic mutant cycle analysis, we recently identified two hydrogen bonds between the amide neck and vanillyl head of capsaicin and T551 (in S4) and E571 (in S4-S5 linker), respectively, of mouse TRPV1. Computational docking to various gating states and interpolated elastic network modeling (iENM) both suggested that the neck-T551 hydrogen bond forms first, followed by formation of the head-E571 hydrogen bond. To test this prediction and reveal the sequential events during capsaicin-induced TRPV1 activation, we carried out a rate-equilibrium linear free energy relationship (LFER) analysis. Residues of the capsaicin-activation machinery are perturbed with a series of point mutations. Single-channel recordings from these mutant channels yielded log-log plots of the opening rate constant versus the equilibrium constant. The slope of this plot, or the phi value, reflects the relative timing of movement by each residue during the closed-to-open transition, with a larger value indicating an earlier event. The phi analysis clearly revealed that the head-E571 hydrogen bond forms after the neck-T551 hydrogen bond. Applying this analysis to residues along the capsaicin-activation machinery will yield a temporal sequence of movements that may offer new insights into the structural mechanism underlying TRPV1 activation by capsaicin.