Biological processes required for life can be understood by studying the underlying molecular and chemical processes which give rise to these biological processes. Proteins are the primary functional molecules of biological systems where they serve as catalysts, cell signaling molecules, receptors and also serve structural and mechanical roles. In order to perform these various functions, proteins must physically interact with other molecules referred to as ligands. Therefore, developing a detailed understanding of protein – ligand interactions from a chemical and molecular perspective improves the understanding of the biological processes which these interactions give rise to. A detailed understanding of these protein ligand interactions also enables the development of drugs capable of disrupting or modulating protein – ligand interactions for the treatment of diseases. In other cases, the study of protein – ligand interactions can be used to develop new and useful biotechnologies. Many techniques can be useful for studying protein – ligand interactions including structural, computational, and biophysical techniques. Of these, biophysical techniques often provide the greatest deal of foundational insight into the nature of the protein – ligand interactions. In addition, a biophysical characterization of a protein – ligand interaction is often a prerequisite for the development of drugs and biotechnological applications for these protein- ligand interactions. Three unique protein – ligand interaction systems are explored in this thesis, using a combination of structural, computational, and biophysical techniques. In the first part of this thesis the interaction between the Laminin N-terminal domain and Ca2+ is explored using a combination of structural, computational, and biophysical techniques. From this approach, a detailed understanding of the molecular basis and functional properties of the interaction between the Laminin N-terminal domain and Ca2+ is developed. In the next part of this thesis, the biophysical and enzymatic characterization of the SARS-CoV-2 Main protease is performed. Guided by this biophysical and enzymatic characterization, an improved substrate for high throughput screening is developed. The final part of this thesis performs a thorough examination of the interaction between Netrin-1 and UNC5B including the requirement for a heparin co-receptor. From this examination of the Nertrin-1 UNC5B interaction, peptide inhibitors of the interaction are rationally designed. Together the work presented in this thesis demonstrates the study of multiple aspects of protein - ligand interactions. It combines studies aimed at gaining an understanding of molecular basis and functional properties of protein – ligand interactions, developing methods to identify and test inhibitors of protein – ligand interactions, and leveraging the gained understanding to rationally design inhibitors of protein- ligand interactions.