Tanacetum parthenium has gained attention for drug development due to its production of bioactive secondary metabolites, such as phenolic compounds (PHEC) and parthenolide (PTN). This work investigated the differences in the production of those compounds among in vitro cultures (organs from plantlets and roots cultures) of T. parthenium. Results showed that the in vitro cultures produced PHEC and PTN, which depended on the type of culture and the incubation time. The content of secondary metabolites was higher in shoots of plantlets (S-ivPL), followed by root cultures (R-ivC) and roots of plantlets (R-ivPL). The PHEC and PTN production was enhanced in plantlets by applying a combination of plant growth regulators (PGRs: α-naphtalenacetic acid [NAA] at 0.27 µM with kinetin [KIN] at 2.32 µM). The highest content of secondary metabolites (62.54 mg gallic acid equivalents g-1; 4.261 and 4.973 mg chlorogenic acid g-1; 0.858 and 1.015 mg caffeic acid g-1; 1.931 mg salicylic acid g-1; 0.071 mg PTN g-1) was found in the PGRs-treated S-ivPL samples. Also, morphogenesis and growth of plantlets was enhanced by these PGRs.