Inositol pyrophosphates (PP-InsPs), which possess functionally significant diphosphate groups, regulate a wide range of biological processes across eukaryotic organisms. The presence and isomeric diversity of PP-InsPs suggest potential biological relevance and enable these molecules to serve as versatile regulators. Identification and characterization of the PP-InsP isomers require specialized approaches. We have successfully utilized X-ray crystallography to directly visualize and identify PP-InsP isomers, thereby informing biological studies and advancing our understanding of their functional roles. Here, we describe detailed methods for identifying PP-InsP isomers in Crystallo.