Bioassay screening or pharmacological evaluation is a common approach to guide the isolation process towards the pure bioactive component. Nevertheless, plenteous time is wasted on isolation, purification and structural elucidation of already known compounds. The tendency over the last years for implementation of high-throughput screening (HTS) technologies leads to the prior identification of the compounds that contribute to the demonstrated activity, avoiding the constant re-isolation of known compounds, reducing workload and cost. The extract of Paeonia mascula ssp. hellenica, which was discriminated for its tyrosinase inhibition among other extracts from Greek flora, was fractionated by FCPC and the resulted fractions were assayed for tyrosinase inhibition potential and further analyzed by HPTLC and NMR. An integrated HPTLC-based procedure for the tracing of compounds that contributed to tyrosinase inhibitory effect in active fractions was established with the use of multivariate data analysis. Additionally, NMR spectral data were correlated with the activity towards tyrosinase resulting in the identification of bioactive compounds through the combination of the Heterocovariance approach (HetCA) and the statistical total correlation spectroscopy (STOCSY). The combined data deriving from NMR and HPTLC correlated to the results of the biological activity by the statistically driven approach, revealed potent whitening agents, providing a major reduction in workload by direct use of routine information.