Prunella vulgaris L. (Labiatae) contains a variety of structurally diverse natural products, primarily rosmarinic acid (RA), ursolic acid (UA) and oleanolic acid (OA), which possess a wide array of biological properties. In the present study, P. vulgaris was harvested at three developmental stages (vegetative, full-flowering and mature-fruiting stages), dissected into stem and leaf tissues and assayed for chemical contents using high performance liquid chromatography. Significant changes in the concentrations of the major secondary metabolites (RA, UA and OA) were observed at the different development stages. The highest concentrations of RA, UA and OA were found at the full-flowering stage (15.83 mg/g dry weight (DW) RA, 1.77 mg/g DW UA and 0.65 mg/g DW OA). Among the different aerial parts of the plant, the concentrations of RA, UA and OA were higher in the leaves than in the stems at the different developmental stages. These results suggest that the full-flowering stage is characterized by the highest concentrations of bioactive compounds. Therefore, this stage may be the optimum point for harvesting P. vulgaris plants. In additional, the leaves of P. vulgaris demonstrated higher RA, UA and OA concentrations than the stems, suggesting higher utilization potential.