We used fluorescence microscopy based quantitative assays in living cells to identify regulators of ER to Golgi trafficking and/or Golgi complex maintenance. We first validated an automated procedure to identify factors, which influence Golgi to ER re-localization of GalT-CFP after BFA addition and wash-out. We then tested 14 proteins that potentially play a role in ER to Golgi trafficking, and localize to the ER and/or Golgi complex when over-expressed. 9 of them interfered with the rate of BFA induced redistribution of GalT-CFP to the ER, 6 of them interfered with GalT-CFP reassembly rate to a juxtanuclear region after BFA wash-out, and 6 of them were positive effectors in both assays. Notably, our live cell approach captures functions of those regulators of ER to Golgi trafficking, which were missed in previous fixed cell assays; as well as assigns respective roles for yet incompletely characterized proteins. Moreover, the assays can be extended to work under the conditions of RNAi and for testing chemical compounds.