This paper provides a novel and robust methodology for determination of nuclear reactor trip setpoints which accounts for uncertainties in input parameters and models, as well as accounting for the variations in operating states that periodically occur. Further it demonstrates that in performing best estimate and uncertainty calculations, it is critical to consider the impact of all fuel channels and instrumentation in the integration of these uncertainties in setpoint determination. This methodology is based on the concept of a true trip setpoint, which is the reactor setpoint that would be required in an ideal situation where all key inputs and plant responses were known, such that during the accident sequence a reactor shutdown will occur which just prevents the acceptance criteria from being exceeded. Since this true value cannot be established, the uncertainties in plant simulations and plant measurements as well as operational variations which lead to time changes in the true value of initial conditions must be considered. This paper presents the general concept used to determine the actuation setpoints considering the uncertainties and changes in initial conditions, and allowing for safety systems instrumentation redundancy. The results demonstrate unique statistical behavior with respect to both fuel and instrumentation uncertainties which has not previously been investigated.