Urban Air Mobility (UAM) is a promising next-generation mobility solution, leveraging airspace and state-of-the-art technologies for efficient and prompt transportation in urban areas. In UAM operations, constrained urban airspace and a high safety requirement level have presented new challenges in determining the separation boundary between aircraft; increasing airspace efficiency while maintaining safety is crucial. In general aviation and Unmanned Aerial Systems (UAS), separation boundaries are set to large values to readily meet a target level of safety (TLS). These large boundaries, however, can severely lower the traffic capacity of UAM airspace if applied directly. To address this issue, in this paper we propose a methodology for determining the safety-assured minimum separation boundary for UAM operations. Our methodology identifies system safety requirements, conducts safety risk assessments (SRA), and analyzes system integrity for a comprehensive systemic approach, which reduces the reliance on extensive flight data without compromising conservatism. We applied the proposed methodology to a Global Navigation Satellite System (GNSS)/Inertial Navigation System (INS) integrated navigation system as a case study for analysis. The results indicate that a significant reduction in separation boundaries is possible while still satisfying the safety requirements compared to existing separation boundaries for general aviation and UAS. Furthermore, this paper applies the proposed method to various navigation technologies such as multi-GNSS constellation, GNSS augmentation systems, and INS sensor with different grades with the goal of further reducing the separation boundary to satisfy the stringent vertical requirements of future UAM airspace.