The US Food and Drug Administration (FDA) has published numerous regulations and guidance documents providing the foundation for establishing the safety of food substances. However, subject matter expertise is needed to interpret regulations and guidance documents commissioned to meet the regulatory standard of reasonable certainty that the substance is not harmful under the conditions of its intended use. To provide guidance for decision-making and communication within the food industry on safety determinations for food substances, a decision tree was developed to inform the user on what approach, supporting information, and documentation are needed. This framework has been developed by industry experts based on considerable experience substantiating safety with a wide range of food substances, to provide a structured approach to help navigate changes that have the potential to impact safety determinations. Several case studies applying the decision tree are presented. The objective of this manuscript is to serve as a reference to promote aligned practices using a decision tree for evaluating food substance safety throughout the food industry, as well as to function as a communication tool between food manufacturers, internal stakeholders, and consumers.