This paper describes a method for quantitatively differentiating crude natural extracts using high-performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI-MS). The method involves performing an HPLC-MS analysis using standard reversed-phase C18 gradient separation on the crude extract. The HPLC system used in this study was a dual-column system designed to optimize throughput. Using image analysis techniques, the data are reduced to a list containing the m/z value and retention time of each ion. The ion lists are then compared in a pairwise fashion to compute a sample similarity index between two samples. The similarity index is based on the number of ions common to both and is scaled from 0 to 1. Extract controls were analyzed throughout a run of 88 unknown fungal extracts. The controls provided information about column and spectrometer stability and overall sensitivity. Pairwise comparison of all control samples indicates that the similarity index is high (0.8) for replicate samples. Comparison between the unknown extract samples produces a distribution of similarities ranging from replicates (0.8) to very dissimilar (0.1). This information can be used to judge the chemical diversity of natural extract samples, which is one approach to determining the quality of libraries being used for drug discovery via high-throughput screening.