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Indiscriminate collection of Arnebia benthamii roots from wild for extraction of red pigments by traders prompted to put this Himalayan plant species under critically endangered category. Concerning the development of sustainable phytoconstituents sources, the present study is aimed at understanding the factors crucial for the scalability of A. benthamii cell suspension cultures. Here, the effect of orbital shaking speed (60–80 rpm) and inoculum density (5–15%) on leaf-induced cell suspension cultures during sequential scale up in shake flask (0.25–5 L) was assessed. The suspension cultures were established on Murashige and Skoog medium supplemented with 10 μM 6-benzylaminopurine and 5 μM indole-3-butyric acid having a 20-days cultivation cycle. The data revealed a significantly (p ≤ 0.05) high biomass productivity (523.80 ± 5.95 g/L, fresh weight) in 0.25 L vessel with 10% inoculum at 70 rpm as compared to large (5.0 L) volume flask (310.80 ± 32.85 g/L). Interestingly, the optimum inoculum density was found to vary with the size of culture vessels. Furthermore, noticeable effects of sequential scaleup with shaking speed and inoculum density were also observed on hydrogen peroxide (stress indicator) production as well as enzymatic (catalase and phenylalanine ammonia-lyase activity) and non-enzymatic (phenolic compounds) antioxidant potential. A. benthamii cells cultivated with optimized conditions had a significant amount of red pigment, with 669.17 ± 26.71 mg dry weight/L yield of total shikonin derivatives. In conclusion, the results clearly demonstrated the likelihood of scalability using optimized process variables for the production of natural red pigments using A. benthamii cell suspension cultures. [ABSTRACT FROM AUTHOR]