We report initial experience in synthesis of (2S,4R)‐4‐[18F]fluoroglutamine, [18F]FGln, which has been used as a tool for monitoring glutamine metabolism in cancer patients. [18F]FGln was prepared by a fully automated PET‐MF‐2V‐IT‐I synthesizer under GMP‐compliant conditions for routine clinical studies. The total radiosynthesis time was about 65 minutes, the decay‐corrected radiochemical yield was 18.0 ± 4.2% (n = 59; failure n = 15), and the radiochemical purity was greater than 90%. In some situations, the yields were low (less than 5%), and the most likely cause of this problem is the initial fluorination step; the fluoride ion might not have been fully activated. In other occasions, low final radiochemical purity was often associated with the failure of the second step—removal of protection groups by anhydrous trifluoroacetic acid. A trace amount of water led to production of undesired 4‐[18F]fluoroglutamic acid. Knowledge learned from the successes and failures of synthesis may be helpful to identify critical steps and pitfalls for preparation of this clinically useful metabolic probe, [18F]FGln, for imaging glutamine utilization in tumor of cancer patients.