Rotationally resolved electronic spectra of three different conformers of 5-methoxytryptamine were recorded in a molecular beam. 5-Methoxy substitution reduces the number of observed conformers to three compared to seven that have been reported for tryptamine. Quantum chemical calculations indicate that anti-rotamers of the methoxy-group are more stable relative to the syn-forms. Two gauche structures (Gpy(in) and Gph(in)) of the ethylamino group with respect to the indole chromophore were found to be less stable than the other seven. The lowest electronically excited state has been identified as the ¹L_b one for all observed conformers which was confirmed by quantum-chemical calculations. Based on the comparison of rotational constants obtained from fits using evolutionary algorithms with those from calculations, the three observed conformers were determined to be the Gpy(up), Gph(up), Gpy(out) ethylamino side-chain conformations.