Motivation: Most data analysis tools for high-throughput screening (HTS) seek to uncover interesting hits for further analysis. They typically assume a low hit rate per plate. Hit rates can be dramatically higher in secondary screening, RNAi screening and in drug sensitivity testing using biologically active drugs. In particular, drug sensitivity testing on primary cells is often based on dose–response experiments, which pose a more stringent requirement for data quality and for intra- and inter-plate variation. Here, we compared common plate normalization and noise-reduction methods, including the B -score and the Loess a local polynomial fit method under high hit-rate scenarios of drug sensitivity testing. We generated simulated 384-well plate HTS datasets, each with 71 plates having a range of 20 (5%) to 160 (42%) hits per plate, with controls placed either at the edge of the plates or in a scattered configuration. Results: We identified 20% (77/384) as the critical hit-rate after which the normalizations started to perform poorly. Results from real drug testing experiments supported this estimation. In particular, the B -score resulted in incorrect normalization of high hit-rate plates, leading to poor data quality, which could be attributed to its dependency on the median polish algorithm. We conclude that a combination of a scattered layout of controls per plate and normalization using a polynomial least squares fit method, such as Loess helps to reduce column, row and edge effects in HTS experiments with high hit-rates and is optimal for generating accurate dose–response curves. Contact: email@example.com Availability and implementation, Supplementary information: R code and Supplementary data are available at Bioinformatics online.