The efficient analysis of small-molecule mixtures underlies many endeavors in chemical biology. The sensitivity of mass spectrometry (MS) has resulted in its widespread adoption for such analyses, and today rapid automated LC-MS analyses are widely used. Several recent studies have demonstrated the feasibility of NMR spectroscopic analyses of complex smallmolecule mixtures, including the use of diffusion-ordered spectroscopy (DOSY) or principal component analysis (PCA) in metabolomics, as well as the characterization of crude unfractionated natural product extracts using routine two-dimensional NMR spectra. Compared to MS analyses, 2D NMR spectroscopic investigations of small-molecule mixtures offer the benefit of more detailed structural information, which is of particular relevance for the detection of novel chemotypes. However, the complexity of 2D spectra typically obtained for small-molecule mixtures has limited a broader implementation of NMR spectroscopy for their characterization. Herein, we describe a simple procedure for the differential analysis of arrays of 2D NMR spectra and demonstrate its utility for the detection of new natural products from a small library of fungal extracts.