Analytical methods play a pivotal role in evaluating high-yielding in vitro cultures for secondary metabolite production. This chapter emphasizes extraction techniques and chemical profiling of these metabolites, discussing techniques like thin-layer chromatography (TLC), gas liquid chromatography (GLC), high-performance liquid chromatography (HPLC), high-performance thin-layer chromatography (HPTLC), and UV spectroscopy for their relevance in phytochemical analysis. Traditional extraction methods such as maceration, percolation, and Soxhlet extraction are commonly used to isolate secondary metabolites from plant tissues. However, modern green extraction techniques like ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and supercritical fluid extraction (SFE) are increasingly preferred due to their reduced environmental impact and enhanced efficiency. Phytochemical screening involves analyzing bioactive compounds in plant extracts. TLC and HPTLC are rapid and cost-effective methods for preliminary screening and separation of compounds based on their polarity. Gas Chromatography (GC) is highly effective for volatile compound analysis, while HPLC offers superior resolution and sensitivity for non-volatile compounds. UV spectroscopy complements these techniques by providing information about the absorbance spectra of specific metabolites. The combination of these analytical methods enables researchers to identify and quantify a wide array of plant-derived bioactive metabolites. These compounds often possess pharmacological activities of significant interest to the pharmaceutical, nutraceutical, and cosmeceutical industries. The integration of conventional and greener extraction techniques with advanced analytical methods enhances the efficiency of screening high-yielding in vitro cultures, facilitating the detection and characterization of bioactive compounds with diverse applications in various fields.