Accurate quantification of substrate characteristics is a central pursuit within the field of surface-enhanced Raman spectroscopy (SERS). A theory based on single-molecule SERS (SM-SERS) statistics was developed for comprehensive substrate evaluation. This approach is applicable to general substrates possessing many hotspots and is capable of quantifying hotspot strength variation using a minimal set of fitting parameters. The model was validated for simulated substrates and then applied to the SM-SERS statistics of a roughened silver electrode, for which the degree of hotspot uniformity was quantified. The fitted model parameters provide important information concerning the structure–activity relationship of hotspots and can be used to directly compare SERS substrates. Overall, our results present an experimentally determinable parameter set that potentially improves upon the widely used “average enhancement factor” metric currently used for SERS substrate evaluation.