SUMMARY: Many natural and designed proteins are only marginally stable limiting their usefulness in research and applications. Recently, we described an automated structure and sequence-based design method, called PROSS, for optimizing protein stability and heterologous expression levels that has since been validated on dozens of proteins. Here, we introduce improvements to the method, workflow and presentation, including more accurate sequence analysis, error handling and automated analysis of the quality of the sequence alignment that is used in design calculations. AVAILABILITY AND IMPLEMENTATION: PROSS2 is freely available for academic use at https://pross.weizmann.ac.il.