Liquid-liquid phase separation (LLPS) is a process that forms membraneless compartments in cells, such as nucleoli and other subcellular structures. This process is increasingly recognized as a key mechanism for organizing cellular functions and is linked to various physiological and pathological conditions, including neurodegenerative diseases and aging. Understanding the biophysical principles and properties of biomolecular condensates is crucial for elucidating their roles in cellular processes. However, the study of LLPS in cells presents challenges, as in vitro observations may not always reflect in vivo conditions. The authors propose guidelines for rigorously characterizing LLPS in vitro and in cells, discuss the limitations of common experimental approaches, and highlight gaps in the field. They emphasize the importance of distinguishing between in vitro and in vivo systems, as well as the need for careful interpretation of phase separation results. LLPS can produce a range of material states, from liquid-like to gel-like or solid-like, and the properties of these states depend on factors such as molecular composition, concentration, and environmental conditions. The ability of proteins and nucleic acids to undergo LLPS is influenced by their sequence and interactions, with intrinsically disordered regions (IDRs) playing a key role. The study of LLPS has important implications for understanding both normal and pathological processes, as well as for developing strategies to manipulate condensates in cellular systems. The authors also discuss the challenges of detecting LLPS in live cells, including the need for accurate metrics and the limitations of current methods. Overall, the study highlights the importance of rigorous experimental approaches and the need for further research to fully understand the role of LLPS in cellular biology.Liquid-liquid phase separation (LLPS) is a process that forms membraneless compartments in cells, such as nucleoli and other subcellular structures. This process is increasingly recognized as a key mechanism for organizing cellular functions and is linked to various physiological and pathological conditions, including neurodegenerative diseases and aging. Understanding the biophysical principles and properties of biomolecular condensates is crucial for elucidating their roles in cellular processes. However, the study of LLPS in cells presents challenges, as in vitro observations may not always reflect in vivo conditions. The authors propose guidelines for rigorously characterizing LLPS in vitro and in cells, discuss the limitations of common experimental approaches, and highlight gaps in the field. They emphasize the importance of distinguishing between in vitro and in vivo systems, as well as the need for careful interpretation of phase separation results. LLPS can produce a range of material states, from liquid-like to gel-like or solid-like, and the properties of these states depend on factors such as molecular composition, concentration, and environmental conditions. The ability of proteins and nucleic acids to undergo LLPS is influenced by their sequence and interactions, with intrinsically disordered regions (IDRs) playing a key role. The study of LLPS has important implications for understanding both normal and pathological processes, as well as for developing strategies to manipulate condensates in cellular systems. The authors also discuss the challenges of detecting LLPS in live cells, including the need for accurate metrics and the limitations of current methods. Overall, the study highlights the importance of rigorous experimental approaches and the need for further research to fully understand the role of LLPS in cellular biology.