AbstractBackground: Alteration in concentrations of blood carnitine and its esters are diagnostic of a number of inherited metabolic disorders. Acylcarnitine (AC) profiles of newborns obtained from dried blood spots by tandem mass spectrometric analysis are being used for the diagnosis of these disorders. There are no data of the postnatal variations of free carnitine (FC) and AC in Indian neonates. Objectives: Evaluation of postnatal variations in free and AC levels in newborns. Methods: Blood FC and AC levels were evaluated in 2,727 healthy neonates of postnatal day 2–30 by electrospray ionization tandem mass spectrometry. Results: Blood C2, C5DC, C16, C16:1, C18, C18:1, C18:2, and C18:OH carnitineswere increased in groups A (aged 8–14 days) and B (aged 15–30 days), compared with the control group (aged 2–7 days), whereas C3, C4, C4OH, C6, C6DC, and C12 carnitines were increased only in group B. No sex‐related differences were found except for C3DC, C4, and C5 carnitine concentrations, which were higher in female neonates. Conclusions: Our data can be used as a reference for the assessment of carnitine status in Indian newborns, hence reducing the risk of misdiagnosis of fatty acid oxidation disorders and organic acidemias during interpretation of the results of tandem mass spectrometry‐based newborn screening. J. Clin. Lab. Anal. 25:126–129, 2011. © 2011 Wiley‐Liss, Inc.