Species of the genus Artemisia mainly biosynthesize sesquiterpene lactones. Achillin is a guaianolide-type sesquiterpene lactone isolated from Artemisia ludoviciana; it has shown antibacterial and anti-inflammatory activities. In addition, achillin exhibits a significant chemosensitizing effect on hepatocellular carcinoma cells resistant to paclitaxel (PTX). The objective of this study was to establish a callus culture from different explants under conditions of light and total darkness to produce achillin. To obtain in vitro cultures, explants of leaves, nodes, internodes, and roots were used, and they were cultured in MS medium with 0.1 mg/L of kinetin (KIN) or benzyl amino purine (BAP) and/or naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA) and 4-amino-3,5,6-trichloro-2-pyridine carboxylic acid (PIC) at 0.1 and 1.0 mg/L. Of all treatments, internodes with BAP (0.1 mg/L) and PIC (1.0 mg/L) grown under photoperiod showed the best friable callus induction, however, GC-MS analysis showed higher achillin content (1703.05 µg/mL) in leaf calluses with PIC (1.0) and KIN (0.1) under photoperiod, and in node plantlets (1880.01 µg/mL) with PIC (0.1) and BAP (0.1). From 12.34 g of dry leaves of Artemisia ludoviciana, 257 mg of achillin were isolated and purified, which was used as a reference in the quantification of achillin in the in vitro culture.