Protein kinase B (PKB/AKT) has been identified as a promising cancer drug target downstream of PI3 kinase. To find novel inhibitors of PKB/AKT kinase activity for progression as anticancer agents, the authors have used a high-throughput screen based on AlphaScreentrade mark technology. A known kinase inhibitor, the isoquinoline H8, was used as a positive control with mean inhibition in the screen of 43.4% +/- 13.1%. The performance of the screen was highly acceptable with Z' and Z factors of 0.83 +/- 0.07 and 0.75 +/- 0.04, respectively. A number of confirmed hits ( approximately 0.1% hit rate) were identified from 63,500 compounds screened. Five compounds have previously been described as PKB inhibitors, demonstrating the ability of the assay to find authentic inhibitors of the enzyme. Five hits had the potential to interfere with the assay signal and were deemed to be false positives. Two compounds were nonspecific inhibitors of PKB as enzyme inhibition in a filter-based assay was markedly reduced in the presence of 0.01% Triton X100. The authors now include an interference assay during hit confirmation procedures and check compound activity in the presence of Triton X100 in an attempt to eliminate nonspecific aggregators at an early stage.