Numerous processes occur simultaneously in the cell both for normal function and in response to changes in the environment. The ability of cells to segregate biochemical reactions into separate compartments is essential to ensure specificity and efficiency in cellular processes. The discovery of liquid-liquid phase separation as a mechanism of compartmentalization has revised our thinking regarding the intracellular organization of molecular pathways such as signal transduction. Here, we highlight recent studies that advance our understanding of how phase separation impacts the organization of biochemical processes, with a particular focus on the tools used to study the functional impact of phase separation. In addition, we offer some of our perspectives on the pathological consequences of dysregulated phase separation in biochemical pathways.