Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality. Artemisinin (ART) and SOMCL-14-221 (221), a spirobicyclic analogue of ART, have been reported to inhibit the proliferation of A549 cells with unclear underlying mechanism. In the present study, we validated that both ART and 221 inhibited the proliferation and migration of NSCLC cells and the growth of A549 xenograft tumors without appreciable toxicity. The proteomic data revealed proteins upregulated in ART and 221 groups were involved in "response to endoplasmic reticulum stress" and "amino acid metabolism". Asparagine synthetase (ASNS) was identified as a key node protein in these processes. Interestingly, knockdown of ASNS improved the antitumor potency of ART and 221 in vitro and in vivo, and treatments with ART and 221 disordered the amino acid metabolism of A549 cells. Moreover, ART and 221 activated ER stress, and inhibition of ER stress abolished the anti-proliferative effects of ART and 221. In conclusion, this study demonstrates that ART and 221 suppress tumor growth by triggering ER stress, and the inhibition of ASNS enhances the antitumor activity of ART and 221, which provides new strategy for drug combination therapy.