Rapid advances in biomedical sciences and genetics have increased the pressure on drug development companies to promptly translate new knowledge into treatments for disease. Impelled by the demand and facilitated by technological progress, the number of compounds evaluated during the initial high-throughput screening (HTS) step of drug discovery process has steadily increased. As a highly automated large-scale process, HTS is prone to systematic error caused by various technological and environmental factors. A number of error correction methods have been designed to reduce the effect of systematic error in experimental HTS (Brideau et al., 2003; Carralot et al., 2012; Kevorkov and Makarenkov, 2005; Makarenkov et al., 2007; Malo et al., 2010). Despite their power to correct systematic error when it is present, the applicability of those methods in practice is limited by the fact that they can potentially introduce a bias when applied to unbiased data. We describe two new methods for eliminating systematic error from HTS data based on a prior knowledge of the error location. This information can be obtained using a specific version of the t-test or of the χ(2) goodness-of-fit test as discussed in Dragiev et al. (2011). We will show that both new methods constitute an important improvement over the standard practice of not correcting for systematic error at all as well as over the B-score correction procedure (Brideau et al., 2003) which is widely used in the modern HTS. We will also suggest a more general data preprocessing framework where the new methods can be applied in combination with the Well Correction procedure (Makarenkov et al., 2007). Such a framework will allow for removing systematic biases affecting all plates of a given screen as well as those relative to some of its individual plates.