<p>The search for sustainable and environmentally friendly production processes drives the development of innovative biotechnological solutions, such as the use of plant cell cultures in controlled bioreactors. These systems are a promising platform for producing high-value bioactive compounds for the pharmaceutical, cosmetic, and agricultural industries. A critical challenge of this technology is the understanding and efficient control of the metabolome to optimize the synthesis of target molecules. In this study, we developed an ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF) method for the comprehensive metabolomic profiling of extracts derived from in vitro cultures of plant cells (<i>Ocimum basilicum</i>). The analytical separation was achieved using reverse-phase chromatography with an optimized solvent gradient. Electrospray ionization (ESI) in both positive and negative modes was applied to maximize analytical coverage. A key feature of this methodology was the utilization of the Zeno trap technology (zenoTOF 7600), which significantly enhanced the sensitivity and improved the metabolite annotation confidence by generating high-quality MS/MS spectra. Data analysis was carried out using MS-DIAL software and subsequent methods to identify and classify the compounds and pinpoint those with significant changes or crucial roles in biosynthetic pathways. This metabolomic approach provides deeper insights into the biosynthesis of active molecules and establishes a robust control strategy for targeted compound production, contributing directly to the advancement of sustainable bioprocesses.</p> Graphical abstract <p></p>