Nardostachys jatamansi (D. Don) DC. (Indian spikenard) belongs to the Caprifoliaceae family and is a medicinal plant that thrives at extreme altitudes in the Himalayan alpine region. It has an array of applications in the field of human healthcare. In this regard, we aimed to enhance the production of two important alkaloids present in N. jatamansi, namely, actinidine and glaziovine, through an in vitro suspension culture treated with biotic and abiotic elicitors at three different concentrations. To induce callus formation in N. jatamansi, leaf explants were inoculated on Murashige and Skoog (MS) basal medium with various combinations of plant growth regulators. A friable callus was observed to have a green colour when treated with 0.2 mg/L of NAA and 0.1 mg/L of BAP, with an induction rate of 31.11 ± 0.5%. Using the same hormonal combination, a suspension culture of N. jatamansi was established in MS liquid medium. Based on the growth curve of the N. jatamansi suspension culture, the elicitors were elicitated at the beginning of the exponential phase and extracted on the 15th day of inoculation. The quantification of both alkaloids was achieved by HPLC. The abiotic elicitor salicylic acid showed the highest actinidine yield 6.95 at 75 µM, whereas the biotic elicitor chitin showed the highest actinidine yield 11.86 at 25 mg/L compared with the control suspension cells. In case of glaziovine production, methyl jasmonate (33.62 at 75 µM) and lipo-polysaccharide 36.18 at 50 mg/L respectively.