Hyptis marrubioides (Lamiaceae) is a medicinal plant that is native from Brazilian Cerrado. In vitro propagation techniques make use of elicitors to alter metabolic pathways, affecting how molecules are produced both qualitatively and quantitatively. This research aimed to evaluate how abiotic elicitors salicylic acid (SA) and silver nitrate (SN) at concentrations of 30µM or 60µM influence Hyptis marrubioides seedling growth by two different in vitro culture methods. The rutin content was quantified by HPLC-DAD. Compared to an untreated culture, the H. marrubioides methanolic extracts cultured in MS medium for 10 days followed by culture in MS medium containing SN (30µM) for 20 days had 1.28 times higher rutin content. In a second experiment, seedlings were cultured in MS medium for 20 days, and then the desired elicitor was added to the culture and allowed to remain in contact with the medium for three and six days. SA (30µM) gave the best results: rutin production was 16.56-foldhigher than the control after six days. SN (30µM) increased the rutin content by 1.17-fold. At the two concentrations evaluated during the elicitation experiments, neither SA nor SN altered the growth parameters shoot length, leaf number, and fresh and dry weight of H. marrubioides seedlings grown in vitro as compared to the control. Based on these results, the abiotic elicitors SA and SN successfully provide Hyptis marrubioides with increased rutin content in vitro.