Phenylketonuria (PKU) is a genetic disorder which can lead to serious long-term complications in children, including intellectual impairment. The cerebral damage can be largely eliminated if hyperphenilalaninemia (HPA) and PKU is detected by newborn screening and the dietary treatment (started no later than 10 days after birth), brings within 15 days the blood Phe level in the ranges 2 -6 mg/dL (or equivalently 120 -360 µmol/L). Consequently, the measurement of plasma Phe level in children with PKU is of utmost importance. We present here in detail two methods for determination of plasma Phe concentrations in children with HPA (detected by newborn screening) or suspected to have PKU based on clinical symptoms. The first method, involving two-dimensional thin-layer chromatography (2D -TLC) on micro scale chromatograms, allows the identification of patients with PKU. However, the quantitation of plasma Phe concentration by the 2D -TLC video-densitometric method is only semiquantitative, because is accompanied