Background & objectives: The reason for lack of data on burden ofHaemophilus influenzae type b (Hib) in developing countries was mainly failure of detection of this fastidious organism in laboratories. Use of isovitalex (IVX) was suggested as an essential supplement for growing this organism. This study was carried out to investigate the impact of 1VX supplementation to chocolate agar for detection of Hib. Methods: Chocolate agar with and without supplementation of IVX was prepared. Clinical samples as well as reference strains of Hib were simultaneously cultured on both the media.