Catharanthus roseus, also known as Madagascar periwinkle, is a perennial plant renowned for its extensive pharmacological properties. It produces vital chemotherapeutic compounds, including vinblastine and vincristine, and exhibits anti‐inflammatory, antidiabetic, and antioxidant activities. In this study, we utilized a range of two‐dimensional (2D) nuclear magnetic resonance (NMR) techniques, such as 1H–1H correlation spectroscopy (COSY), 1H–1H J‐resolved NMR, and 1H–13C heteronuclear single quantum coherence (HSQC) sensitivity‐enhanced NMR spectroscopy, to identify key metabolites in C. roseus leaf extracts. Given the presence of numerous metabolites with closely spaced multiplet resonances, the 1H NMR spectra often exhibit significant signal overlap, making metabolite identification difficult or even impossible. However, the use of 2D NMR techniques effectively overcame this challenge, allowing for the precise identification of important alkaloids, such as vindoline, vinblastine, serpentine, and ajmalicine, along with essential metabolites like organic acids, amino acids, and carbohydrates. The extract contained a variety of bioactive compounds, including organic acids crucial for the tricarboxylic acid (TCA) cycle, branched‐chain amino acids vital for metabolic functions, and alkaloids with substantial therapeutic potential. This comprehensive study underscores the continued significance of C. roseus in both traditional and modern medicine, emphasizing its intricate metabolic network and its potential in the development of novel therapeutics.