A liquid chromatography--mass spectrometry (LC-MS) method with selected ion monitoring (SIM) was developed and validated for the analysis and standardization of artemisinin in Artemisia annua L. This method is simple and accurate and requires only an 11 min per sample running time for the direct detection and quantification of artemisinin, a sesquiterpene lactone with antimalarial activity. To accurately analyze artemisinin, SIM was used to record the abundance of the [M - 18 + H]+ ion peak at m/z 265.3, with a scan range between m/z 250 and 270. Quantification was based on the LC-MS peak area of artemisinin, and the standard curve was used for calculation. This method was then validated and applied to several populations of A. annua to assess the population means and the diversity within a selected population. The distribution of artemisinin was found to vary by plant origin with population means ranging from 0.03 to 0.71% artemisinin dry leaf weight. Individual plants within the selected population that exhibited the highest artemisinin mean were then sampled, and the contents ranged from trace amounts to 1.5% artemisinin dry leaf weight basis, making these single plants accumulating artemisinin in concentrations >1.0% promising candidates for plant breeding and varietal development for high-yielding artemisinin.