Background: Phenylketonuria is an autosomal recessive condition in which phenylalanine accumulates in the blood and bodily fluids due to a lack of Phehydroxylase activity. The study aimed to evaluate the effect of instructional guidelines on mothers' knowledge, practice and lifestyle of their children with phenylketonuria. Design: A quasiexperimental research design was utilized to achieve the study's aim. Setting: The study was conducted at genetic counseling clinic in the basic health care unit at Sohag City affiliated to the Ministry of Health (MOH). Sample: A purposive sample included 100 mothers accompanied by their children with Phenylketonuria. Tools: Two tools were used: Tool I: Mothers' knowledge and reported practice regarding phenylketonuria: a structured interview questionnaire was utilized and Tool II: Children's lifestyle assessment questionnaire. Results: The study revealed that there was a statistically significant improvement in post-intervention mothers' knowledge and reported practices and their children's lifestyle with phenylketonuria than pre-intervention. A statistically significant correlation detected between compliance with the prescribed diet and growth problems. No statistically significant correlation found between compliance with the prescribed diet and sleep problems. Conclusion: The study concluded that the instructional guidelines were effective in improving mothers' knowledge, reported practice, and their children's lifestyle with phenylketonuria. Recommendations: Health education program for mothers should be provided to increase their knowledge and reported practices about phenylketonuria, as well as identify the importance of a prescribed diet for children with this condition.