• Lichen secondary metabolites show important biological activities as well as pharmaceutical and chemotaxonomic potential. In order to utilize such substances of interest, detailed knowledge of their biosynthetic pathways is essential. • 13CO₂-pulse/chase experiments using intact thalli of the lichen Usnea dasopoga resulted in multiple 13C-labeled isotopologs in amino acids, but not in the dibenzofuran derivative usnic acid – one of the best-studied lichen metabolites, with considerable and renewed interest for pharmaceutical and lifestyle applications. • Spraying an aqueous solution of U-13C₆glucose onto the thalli of U. dasopoga afforded a specific mixture of multiple 13C-labeled isotopologs in usnic acid. One- and two-dimensional NMR analysis of the crude lichen extract corroborated the polyketide biosynthetic pathway via methylphloroacetophenone but not via phloroacetophenone. • With usnic acid as an exemplar, we provide proof-of-principle experiments that can be used in general to study metabolic pathways and fluxes in intact lichens.