Hydrogen peroxide (H₂O₂), a reactive oxygen specie (ROS), is most commonly associated with oxidative stress causing cytotoxic effects on living cells. Oxidative stress has been implicated in various conditions including neurodegenerative diseases, autoimmune diseases and cancer. In addition H₂O₂ is produced as a defense mechanism against pathogens, as being released by activated phagocytes. In recent years, H₂O₂ has become established as an important regulator of signal transduction in eukaryotic cells. Hydrogen peroxide is generated both intracellularly and extracellularly in response to various stimuli including cytokines and growth factors. There are different mechanisms by which H₂O₂ is generated, facilitating signal transduction in cells; through NOX-system in miyochondria, via singlet oxygen, receptor/ligand interaction or by redox active metal ions. The HIV glycoprotein 120 (gp120) is associated with HIV dementia and it is known as a neurotoxin that causes neuronal damage. It has been proposed that free radicals may be involved in the pathogenesis caused by gp120. In addition the truncated form of thioredoxin (Trx80) is known to stimulate HIV replication in HIV infected cells, however, the exact mechanism is not known. A possible way both proteins may mediate their activity is by inducing H₂O₂ production. The aim of this study was to investigate H₂O₂ production induced by the proteins gp120 and Trx80. In order to detect H₂O₂ production an assay based on the fluorescent compound Amplex Red, was established. The assay was used to detect H₂O₂ released by gp120 and Trx80 in a cell-free environment, in a cell-system and in the presence of metal ions (copper ions) with a physiological reductant (ascorbate). We did not detect H₂O₂ production induced by gp120 and Trx80 respectively, using our assay, however, other ROS such as hydroxyl radicals may have been generated although they were not detectable with our method. Hence, further studies are needed in order to fully understand how gp120 and Trx80 mediate their activity.