Double-mutant cycle (DMC) analysis is a powerful approach for detecting and quantifying the energetics of both direct and long-range interactions in proteins and other chemical systems. It can also be used to unravel higher-order interactions (e.g. three-body effects) that lead to cooperativity in protein folding and function. In this review, we describe new applications of DMC analysis based on advances in native mass spectrometry and high-throughput methods such as next generation sequencing and protein complementation assays. These developments have facilitated carrying out high throughput DMC analysis, which can be used to characterize increasingly higher-order interactions and very large interaction networks in proteins. Such studies have provided insights into the extent of cooperativity (epistasis) in protein structures. High-throughput DMC studies have also been used to validate correlated mutation analysis and can provide restraints for protein docking.