To support the development and evaluation of future function allocation concepts for separation assurance systems for the Next Generation Air Transportation System, this paper presents the design and human-in-the-loop evaluation of three feasible function allocation concepts that allocate primary aircraft separation assurance responsibilities and workload to: 1) pilots; 2) air traffic controllers (ATC); and 3) automation. The design of these concepts also included rules of the road, separation assurance burdens for aircraft of different equipage levels, and utilization of advanced weather displays paired with advanced conflict detection and resolution automation. Results of the human-in-theloop simulation show that: a) all the concepts are robust with respect to weather perturbation; b) concept 1 (pilots) had highest throughput, closest to assigned spacing, and fewest violations of speed and altitude restrictions; c) the energy of the aircraft during the descent phase was better managed in concepts 1 and 2 (pilots and ATC) than in concept 3 (automation), in which the situation awareness of pilots and controllers was lowest, and workload of pilots was highest. The paper also discusses further development of these concepts and their augmentation and integration with future air traffic management tools and systems that are being considered for NextGen.