Conventional hydropower potential assessment (HPA) does not explicitly account for operational requirements and constraints affecting hydropower availability in multifunctional river systems (MRSs). This study introduces Operational Hydropower Potential Assessment (OHPA), a hierarchical framework integrating hydrological conditions, operational requirements, and constraints into conventional HPA. The framework distinguishes hydrological assessment from operational assessment and combines their outcomes to determine reference and operational hydropower potential (OHP). The methodology was applied to five operational reaches of the upper canalised Vistula River in Poland using hydrological observations, technical hydropower parameters, and documented operational information. Daily water discharge exhibited strong spatial coherence, with Spearman correlations of 0.815–0.972 and the first principal component explaining 92.38% of total variance. Annual hydro-technical reference energy ranged from 66.90 to 79.01 GWh. For the Smolice–Łączany reach, the OHPA index decreased from 0.752 to 0.390 under a very-low-flow scenario, demonstrating the sensitivity of hydropower availability to hydrological extremes. Documented high-water closures occurred near the 99th percentile of long-term discharge, highlighting additional operational limitations. OHPA provides a transferable framework for quantifying hydropower availability under variable hydrological conditions, operational constraints, and competing water uses in multifunctional river systems.