Background and Objective: Centaurea pumilio L. (Asteraceae) is a wild plant rich in valuable secondary metabolites. The aim of this study was the induction and maintenance of callus culture in addition to, the analysis of secondary metabolites in the in vitro induced tissues. Biotic and abiotic elicitors were used to enhance the production of flavonoids and phenolics in the callus. Material and methods: Calli were induced from seedling explants cultured on Murashige and Skoog solid media (M&S) supplied with different combinations of phytohormones. Callus growth parameters besides its productivity of flavonoids and phenolics were monitored for each treatment. M&S media supplied with 1 mg/L naphthalene acetic acid and 1 mg/L benzyl amino purine showed the maximum growth parameters while, media with 1 mg/L naphthalene acetic acid and 1 mg/L kinetin exhibited the maximum flavonoids and phenolics productivity compared with other media. Different elicitors of biotic and abiotic origin were tried to investigate their effect on increasing productivity of flavonoids and phenolic contents. Results: The best applied elicitor was yeast elicitor and the highest productivity of both flavonoids and phenolics was observed at 6th day after elicitor addition. Moreover, aqueous extract of Spirulina platensis added to M&S media as a substitute for phyto hormones showed an increase in callus growth parameters, flavonoids and phenolic contents. Thirty-two secondary metabolites were tentatively identified in callus grown on M&S with 1 mg/l naphthalene acetic acid and 1 mg/l kinetin using UPLC-ESI-MS/MS technique in positive and negative ionization mode. Conclusion: M&S media supplemented with 1 mg/L naphthalene acetic acid and 1 mg/L benzyl amino purine was used to obtain the best callus vegetative growth. While the maximum phenolics and flavonoids production was obtained from callus grown on M&S media supplemented with 1 mg/L naphthalene acetic acid, 1 mg/L kinetin and 12 mg/L yeast extract as a biotic elicitor.