14-3-314-3-3 proteins are a conserved, widely expressed, and abundant family of acidic polypeptides that recognize phosphoserine (SEP) and phosphothreonine (TPO)Phosphothreonine motifs on the target proteins. They have attracted considerable interest due to their essential roles in cellular signaling pathways. Quantifying ligand-induced conformational changes is critical for elucidating their functions. Without a clear understanding of these conformational change mechanisms, structure-based drug and protein design remains a major challenge. The Triangular Spatial Relationship (TSR)-basedTriangular spatial relationship method is a computational approach developed by the authors of this chapter for comparing molecular 3D structures and investigating molecular interactions. In this chapter, we aim to illustrate the utility of the TSR-based method in defining and quantifying ligand-induced conformational changes in 14-3-3 isoforms using experimentally resolved 3D structures. The key contributions of this work are as follows: (i) Introduction of a new 14-3-3 dataset enriched with biologically meaningful annotations and labels. (ii) Application of hierarchical clustering and multidimensional scalingMultidimensional scaling to reveal distinct structural features and diversity among 14-3-3 isoforms. (iii) Demonstration thatTSR keys TSR keys-based analysis effectively represents and quantifies conformational changes of 14-3-314-3-3σ upon binding to SEP or TPO residues on target proteins. (iv) Evidence that TSRTriangular spatial relationship keysTSR keys designed to capture the 3D geometry of amino acids can distinguish arginine residues within the SEP or TPO binding sites of 14-3-3σ from those outside these regions. In summary, this study demonstrates the effectiveness of an advanced computational framework for characterizing ligand-induced conformational changes in 14-3-3 isoforms of both backbone and side chain geometries.