Introduction: Iris L. are promising in medicine due to the biological activity of extracts. Iris sibirica L. is spread in Russia but its phytochemical composition has not been studied in detail though it is included in the Red Book. For this reason, I. sibirica L. biotechnology is in high demand. One of the key points in biotechnology is the regulation of plant metabolism using phytohormones. Obtaining of chromatographic metabolite profiles allows to control this process. Objective: The aim of this study was to develop an approach for effective control of biotechnological raw materials of I. sibirica L. by flavonoid profiles using high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) and to investigate the influence of phytohormones in nutrient media on content of flavonoids. Methodology: Iris sibirica L. regenerated plants were grown on Murashige–Skoog media with 6-benzylaminopurine (6-BAP) and α-naphtylacetic acid (NAA) additives. To optimise extraction conditions, the design of the experiment was used. Profiles of polyphenols were obtained by HPLC–MS/MS in the positive and negative ionisation modes. Results: The process for efficient extraction from leaves of I. sibirica L. were developed. The factors influencing the extraction efficiency of flavonoids have been determined. A total of 36 compounds were identified by HPLC–MS/MS. Among them isoflavones and their glycosides are the main classes. Addition of an auxin-like hormone increased the non-polar flavonoid levels, but decreased the polar ones. The variation in concentration of cytokinin (6-BAP) affected almost all of the analytes. Conclusion: The methodology for effective control of I. sibirica L. raw plant material biotechnology was developed by analysing obtained chromatographic polyphenol profiles.