Reduced oxygen supply that does not satisfy tissue and cellular demand (hypoxia) regularly occurs both in health and disease. Hence, the capacity for cellular oxygen sensing is of vital importance for each cell to be able to alter its energy metabolism and promote adaptation to hypoxia. The hypoxia-inducible factor (HIF) prolyl hydroxylases 1-3 (PHD1-3) and the asparagine hydroxylase factor-inhibiting HIF (FIH) are the primary cellular oxygen sensors, which confer cellular oxygen-dependent sensitivity upon HIF as well as other hypoxia-sensitive pathways, such as nuclear factor κB (NF-κB). Studying these enzymes allows us to understand the oxygen-dependent regulation of cellular processes and has led to the development of several putative novel therapeutics, which are currently in clinical trials for the treatment of anemia associated with kidney disease. Pharmacologic inhibition and genetic knockdown are commonly established techniques in protein biochemistry and are used to investigate the activity and function of proteins. Here, we describe specific protocols for the knockdown and inhibition of the HIF prolyl hydroxylases 1-3 (PHD1-3) and the asparagine hydroxylase factor-inhibiting HIF (FIH) using RNA interference (RNAi) and hydroxylase inhibitors, respectively. These techniques are essential tools for the analysis of the function of the HIF hydroxylases, allowing the investigation and discovery of novel functions and substrates of these enzymes.