To investigate the longitudinal changes in amino acid (AA) and acylcarnitine (AC) profiles of preterm neonates over the first 2 wk of life, and to detect any significant deviation from full term values that requires change of cut-off values used for detection of metabolic disorders in preterm neonates. This observational analytical longitudinal study was conducted on 131 premature neonates (gestational age ranged from 27 to 36 wk) and 143 healthy full-term neonates. Dried blood spots were taken on the 5th and 14th postnatal day from the premature neonates and on day 5 from full term neonates for neonatal screening. Samples were analyzed for AA and AC using tandem mass spectrometer. Most AA significantly decreased on day 14 compared to day 5 among preterm neonates (p < 0.05). The combined values of total carnitine (TC), total acylcarnitine (tAC) and short-chain acylcarnitines on day 5 among preterm neonates were statistically significantly higher compared to the day 14 sample (p 0.0001), whereas no statistically significant difference was found regarding the values of medium-, long-chain acylcarnitines, tAC/FC, and FC/TC (p > 0.05). The levels of AA of preterm neonates were statistically significantly higher than that of the controls (p < 0.05). The values of TC, tAC, short-, medium- and long-chain acylcarnitines, were significantly higher than those of the controls (p < 0.05). The reference ranges for preterm neonates were determined using the 1st and 99.9th percentiles. AA and AC showed an age-related distribution of their concentrations. This underlines the importance of using appropriate reference values when working with a prematurely born population.