Conventional isolation methods in natural products chemistry are time-consuming and costly and often result in the isolation of moderately active compounds or the detection of already known natural products (NPs). A fast and cost-effective way to identify bioactive metabolites in plant extracts prior to isolation has been developed based on the nuclear magnetic resonance (NMR)-heterocovariance approach (NMR-HetCA). In order to evaluate in depth the application of this chemometrics-based drug discovery methodology, simple mixtures of 10 standard NPs simulating a fast centrifugal partition chromatography (FCPC) fractionation (artificial fractions, ArtFrcts), as well as a more complex mixture of 59 natural standard substances simulating a crude plant extract (artificial extract, ArtExtr), were prepared. FCPC was employed for the fractionation of the ArtExtr, while the inhibitory activity of all fractions against DPPH was evaluated, and their chemical profile was recorded using NMR spectroscopy. Spectral information was processed in the MATLAB environment, and statistical approaches, including HetCA and statistical total correlation spectroscopy (STOCSY), were applied to identify bioactive compounds. Total heterocovariance plots (pseudospectra) facilitated the detection of highly correlated metabolites and led to the direct identification of 52.6% of the active compounds. The success in identifying the ArtExtr bioactive substances increased to 63.2% when spectral alignment was implemented. HetCA incorporates chromatographic (fractionation), spectroscopic (NMR profiling), and bioactivity results along with advanced chemometrics and could be established as a method of choice for the rapid and effective identification of bioactive NPs in plant extracts prior to isolation.