The rising demand for picrosides commercially and over-exploitation ofPicrorhiza kurroafrom natural habitat has to initiate alternative strategies for sustainable production of metabolites. In the present research, wild leaf explant ofP. kurroawas used to produce friable callus under different culture condition, i.e., dark and light with two temperature variants (15 degrees C and 25 degrees C). Afterward, callus cell lines were screened based on growth biomass and metabolites content accumulation. The results revealed, maximum callus growth index along with antioxidant potential (IC50-40.88 mu g/mL) and total phenol content (41.35 mu g/mg) were observed under dark 25 degrees C. However, under light 15 degrees C, highest accumulation of picroside II (0.58 mu g/mg), cinnamic acid (0.15 mu g/mg),p-hydroxy acetophenone (0.30 mu g/mg), total flavonoids (77.30 mu g/mg), nitrogen (7.06%), carbohydrates (18.03%), and protein (44.12%) were detected. Major reported metabolite in callus was picroside I (1.63 mu g/mg) under dark 15 degrees C. For the first time, picroside III content (range 0.15-0.56 mu g/mg) was also detected and quantified in leaf-derived calli. Expression profiling of picroside biosynthetic pathway genes showed a positive correlation with the observed metabolites. Furthermore, an optimized protocol of metabolites enriched callus biomass could be used as potential strategy for sustainable production of picrosides at commercial scale.