Many genetic (gene deletion, interruption or mutation), epigenetic (such as antisense or small interfering RNA) and immunological methods are being applied in 'high-throughput target validation' studies of the novel potential targets arising from whole genome sequencing. Such applications often focus on 'loss of function' approaches. However, target validation is most reliable when multiple orthogonal approaches are used. Initiating a target-based discovery project based on correlative evidence is faster than awaiting causative evidence. Indeed, the multiple tools needed to generate firm proof usually include methods and reagents only generated after starting a discovery project with little evidence beyond correlations. Robust and rigorous tests of whether a drug candidate is efficacious in vivo because of its effects on a specific molecular particular target are best made by simultaneously applying multiple orthogonal tools. Examples of the orthogonal tools approach will be discussed.