A number of technologies, including CRISPR/Cas, transcription activator-like effector nucleases and zinc-finger nucleases, allow the user to target a chosen locus for genome editing or regulatory interference. Specificity, however, is a major problem, and the targeted locus must be chosen with care to avoid inadvertently affecting other loci (‘off-targets’) in the genome. To address this we have created ‘Genome Target Scan’ (GT-Scan), a flexible web-based tool that ranks all potential targets in a user-selected region of a genome in terms of how many off-targets they have. GT-Scan gives the user flexibility to define the desired characteristics of targets and off-targets via a simple ‘target rule’, and its interactive output allows detailed inspection of each of the most promising candidate targets. GT-Scan can be used to identify optimal targets for CRISPR/Cas systems, but its flexibility gives it potential to be adapted to other genome-targeting technologies as well.