Fragmentation by artificial barriers is an important threat to freshwater biodiversity. Mitigating the negative aftermaths of fragmentation is of crucial importance, and it is now essential for environmental managers to benefit from a precise estimate of the individual impact of weirs and dams on river connectivity. Although the indirect monitoring of fragmentation using molecular data constitutes a promising approach, it is plagued with several constraints preventing a standardized quantification of barrier effects. Indeed, observed levels of genetic differentiation GD depend on both the age of the obstacle and the effective size of the populations it separates, making comparisons of the actual barrier effect of different obstacles difficult. Here, we developed a standardized genetic index of fragmentation (FINDEX), allowing an absolute and independent assessment of the individual effects of obstacles on connectivity. The FINDEX is the standardized ratio between the observed GD between pairs of populations located on either side of an obstacle and the GD expected if this obstacle completely prevented gene flow. The expected GD is calculated from simulations taking into account two parameters: the number of generations since barrier creation and the expected heterozygosity of the populations, a proxy for effective population size. Using both simulated and empirical datasets, we explored the validity and the limits of the FINDEX. We demonstrated that it allows quantifying effects of fragmentation only from a few generations after barrier creation and provides valid comparisons among obstacles of different ages and populations (or species) of different effective sizes. The FINDEX requires a minimum amount of fieldwork and genotypic data and solves some of the difficulties inherent to the study of artificial fragmentation in rivers and potentially in other ecosystems. This makes the FINDEX promising to support the management of freshwater species affected by barriers, notably for planning and evaluating restoration programs.