Perezone, a sesquiterpenic benzoquinone with diverse medicinal properties, accumulates in the roots of Acourtia species. In this time-dependent study, the production of perezone was followed in A. cordata culture systems of plants kept in vitro or acclimated and grown in pots. Perezone was characterized by several analytical methods, using the crystallized compound isolated from roots of wild plants as standard. A procedure was developed for its selective quantification, which considers the specific bathochromic shift of the absorbance band in the visible region between the spectra of perezone in its non-ionic and ionic forms, with intensity directly proportional to concentration. In vitro, perezone was recovered from A. cordata roots in average amounts of 5.21 mg g−1 dry weight. Contrastingly, in plants under ex vitro conditions, perezone in roots increased logarithmically, rising from an average of 2.4 mg g−1 dry weight at the 12th week, to 43.6 mg g−1 dry weight at the 31st week, an amount comparable to wild plants. These findings show the feasibility of in vitro and ex vitro culture systems to propagate and conserve the germplasm of perezone-producing Acourtia plants, and a fast and reliable method for the quantification of this valuable compound.