The NEDD8 conjugation pathway is initiated by the NEDD8 E1, also known as NEDD8 activating enzyme (NAE) or APPBP1/UBA3 (Gong, Yeh. J Biol Chem 274:12063-12042, 1999). The best described biological role for NEDD8 conjugation is to regulate the activity of the cullin RING ligase (CRL) family of ubiquitin E3 ligases (Gong, Yeh. J Biol Chem 274:12063-12042, 1999). In this way, the NEDD8 pathway regulates the turnover of a subset of ubiquitin proteasome system (UPS) substrates that are essential for cancer cell growth and survival (Soucy, Smith, Milhollen. Nature 458:732-737, 2009). We recently initiated clinical trials with a first-in-class small molecule inhibitor of NAE for the treatment of cancer (Soucy, Smith, Milhollen. Nature 458:732-737, 2009). Here we describe a biochemical and cell-based assay used to identify NAE inhibitors and monitor inhibition of the NEDD8 conjugation pathway.