Artemisia annua is well known for biosynthesizing artemisinin, which is the primary therapeutic approach against malaria. It was reported that treatment with leaf of A. annua showed better effect and less tendency of developing drug resistance than purified artemisinin, suggesting other components in A. annua may contribute to the therapeutic efficacy. Here, we conducted a global proteomic profiling of A. annua with identification of a total of 13,403 proteins based on the genome sequence annotation database. Furthermore, we generated a spectral library to perform quantitative proteomic analysis using data independent acquisition mass spectrometry (DIA-MS). Specifically, we comprehensively quantified and compared proteins between two chemotypes that produce high (HAP) and low (LAP) artemisinin content, respectively. 182 proteins were identified with abundance significantly different between these two chemotypes. Overall, our current study globally identified the proteome of A. annua and quantitatively compared the targeted sub-proteomes between the two cultivars of HAP and LAP, providing systematic information on metabolic pathways of A. annua and facilitating identification of good chemotypes for producing anti-malaria compounds.