This study was conducted to investigate the influence of Copper sulfate (CuSO₄) and Cobalt chloride (CoCl₂) on callus biomass production, total phenolics accumulation and antioxidant activity of Artemisia annua. The in vitro leaf explants were cultured on Murashige and Skoog (MS) medium contained 1 mg/l α-naphthalene acetic acid (NAA) and 0.5 mg/l 6-benzyl adenine (BA) for callus induction. CuSO₄ and CoCl₂ were added separately in four concentrations (1, 2, 5 and 10 mg/l) to MS medium. CuSO₄ at 2 mg/l was the most appropriate level for callus fresh and dry weights which recorded 1.29 g/explant and 0.122 g/explant, respectively. Application of CoCl₂ at different levels showed a positive impact on the growth of callus. Herein, the highest callus fresh weight (1.44 g/explant) and dry weight (0.139 g/explant) were achieved by 2 mg/l CoCl₂, when compared with control (0.97 g/explant for fresh weight and 0.092 g/explant for dry weight). Elicitation with CuSO₄ at 2 mg/l recorded the highest total phenolic production (8.75 mg/g dry weight (DW)). However, the highest significant percentage of antioxidant activity (90.85%) was recorded for 5 mg/l CuSO₄-elicited culture. For CoCl₂, the highest significant values of total phenolic content (8.40 mg/g DW) and antioxidant activity (91.46 %) were achieved by 2 mg/l CoCl₂ enriched medium compared to control (6.66 mg/g DW and 90.68%, respectively). The current study revealed the possibility of using callus cultures of A. annua as a promising tool for the in vitro production of phytochemicals.