Site-directed mutagenesis is routinely used in modern biology to elucidate the functional or biophysical roles of protein residues, and plays an important role in the field of rational protein design. Over the past decade, a number of computational tools have been developed that can predict the effect of point mutations on a protein's biophysical characteristics. However, these programs usually provide predictions for only a single characteristic. Furthermore, online versions of these tools are often impractical to use for examination of large and diverse sets of mutants. We have created a new web application, (http://enzyme.ucd.ie/PEAT_SA), that can simultaneously predict the effect of mutations on stability, ligand affinity and pK a values. PEAT-SA also provides an expanded feature-set with respect to other online tools which includes the ability to obtain predictions for multiple mutants in one submission. As a result, researchers who use site-directed mutagenesis can access state-of-the-art protein design methods with a fraction of the effort previously required. The results of benchmarking PEAT-SA on standard test-sets demonstrate that its accuracy for all three prediction types compares well to currently available tools. We illustrate PEAT-SA's potential by using it to investigate the influence of mutations on the activity of Subtilisin BPN′. This example demonstrates how the ability to obtain a wide range of information from one source, that can be combined to obtain deeper insight into the influence of mutations, makes PEAT-SA a valuable service to both experimental and computational biologists. Proteins 2010. © 2010 Wiley-Liss, Inc.