Phenylketonuria (PKU, OMIM #261600) is a classic example of an inborn error of metabolism, treatable with careful dietary management with a good clinical outcome. Classical PKU is caused by the deficiency of phenylalanine hydroxylase (PAH, EC 1.14.16.1) resulting in the accumulation of excess phenylalanine (Phe) from dietary protein and causing the condition of hyperphenylalaninaemia (HPA), which is the main cause of neurological disturbances and intellectual disability in cases of untreated PKU. The present study used two different methods for Phe measurement from DBS which includes Labsystems neonatal phenylalanine kit and an LC-MS/MS-based method. Both the methods were regularly evaluated for internal and external quality controls which exhibited a good correlation.