Background Neonatal feeding intolerance may be the presentation of many diseases. Response to enteral feeding depends on enterocyte mass and function. Objective The aim was to assess the diagnostic and prognostic role of plasma citrulline level as a noninvasive marker in neonatal feeding intolerance. Patients and methods Thirty-nine neonates from the neonatal intensive care were classified into two groups: the patients’ group which included 26 neonates with different diagnosis, all of them had enteral feeding intolerance and the control group which included 26 neonates of similar gestational age and birth weight with no feeding intolerance. Patients with significant renal insufficiency or suspected urea cycle defect were excluded. This is a hybrid study. It was carried out in two phase. Phase 1: the case–control study in which we measured plasma citrulline in the patients’ and control group at the start of the study; and phase 2 the cohort study in which followup of plasma citrulline was done in patients at the time of diagnosis of feeding intolerance, when they started feeding, and when they reached 50% of their requirements. Results Plasma citrulline level at day 1 of presentation was significantly lower in the patients’ group than in the control group. In the absence of intestinal injury, plasma citrulline level increased significantly with the increase in gestational age, but once intestinal injury for any cause occurred, plasma citrulline level was no more age dependent. With more oral feeding tolerance, the plasma citrulline level increased significantly. Conclusion Plasma citrulline level is a very good noninvasive marker of intestinal integrity; it can assess the readiness for enteral feeding and it is not disease specific (whether medical or surgical conditions).