Factor Inhibiting HIF (FIH) is an asparaginyl hydroxylase which uses oxygen to hydroxylate target proteins, such as the C-terminal transactivation domain of Hypoxia Inducible Factor-α (HIFCAD). FIH hydroxylation of the HIFCAD inhibits the transcriptional activity of HIF transcription factors, downregulating the expression of genes required for adaption to hypoxia. Physiologically, FIH is also a key regulator of metabolism, with FIH knockout mice displaying improved insulin sensitivity and protection against weight gain. However, FIH has not yet been targeted therapeutically and all current inhibitors are non-specific or lack potency. To overcome this, we developed a robust, stable and adaptable cell-based dual fluorescent reporter system suitable for high throughput small molecule screening to identify novel FIH inhibitors. Expression of a gal4DBD-HIFCAD synthetic transcription factor is paired with expression of a dual fluorescent reporter, where one fluorescent gene is FIH-regulated and the second is constitutively expressed, acting as an internal control. Applicable to high content imaging and FACS-based screening, this system achieves 11-fold reporter induction above background in over 97% of the cell population upon FIH inhibition and is highly robust with clear signal separation (Z’=0.68). FIH-specificity has been verified through the generation of a FIH knockout reporter line and the system has been successfully scaled down to a 1536-well plate format, confirming suitability for high throughput screening. For counter screening purposes (i.e. identification of autofluroescent compounds), we have generated a stable NanoLuciferase-based reporter, termed NanoFIRE, which is also suitable for screening under hypoxic conditions and can be adapted to investigate other endogenous transcription factors, including HIF and the Progesterone Receptor. The FIH dual fluorescent reporter system is currently being used in a 300,000-compound small molecule screen at the National Drug Discovery Centre (WEHI), with the aim to identify novel FIH inhibitors with potential future applications in the treatment of metabolic disease.