Finally, and remarkably, non-covalent interaction network analysis appears to be able to predict ~50% of hotspots in both our designed Kemp eliminase as well as two naturally evolved proteins. Also, we demonstrate that further evaluation based on evolutionary conservation of interaction networks not only rationalizes experimental engineering outcomes but also acts as a tool to filter viable vs. non-viable hotspot residue predictions from our interaction network analysis. Taken together, these tools provide a fully computational alternative to NMR for hotspot prediction for systems where NMR analysis is not viable.