Oxygen homeostasis determines the activity and expression of a multitude of cellular proteins and the interplay of pathways that affect crucial cellular processes for development, physiology, and pathophysiology. Hypoxia-inducible factors (HIFs) are transcription factors that respond to changes in available oxygen in the cellular environment and drives cellular adaptation to such conditions. Selective gene expression under hypoxic conditions is the result of an exquisite regulation of HIF, from the pre-transcriptional stage of the HIF gene to the final transcriptional activity of HIF protein. We provide a dissected analysis of HIF modulation with special focus on hypoxic conditions and HIF pharmacological interventions that can guide the application of any future HIF-mediated therapy.