Liquid Chromatography tandem mass spectrometry (LC-MS/MS) is used for the diagnosis of more than 30 inborn errors of metabolisms (IEMs). This study aimed to (1) determine the age-specific cut-off values of AAs and ACs in Bangladesh and (2) validate the LC-MS/MS method for diagnosis of the patients with IEMs. For this, quantitation of AAs and ACs was performed on an automated LC-MS/MS system using a Neo-Mass AAAC kit (Labsystems Diagnostics Oy, Finland) from dried blood spot (DBS) cards. The method validation data clearly indicated that this method was suitable for the quantitation of AA and AC from the DBS specimens. Then second-tier test for confirmation of the screening positive cases was conducted by urinary metabolic profiling using gas chromatography-mass spectrometry (GC-MS). Out of 9 cases that came out as screening positive by LC-MS/MS, seven cases were confirmed by urinary GC-MS analysis including 3 cases with phenylketonuria, 1 with citrullinemia type II, 1 with methylmalonic acidemia, 1 with isovaleric acidemia and 1 with carnitine uptake defect. Two borderline positive cases with MCAD were found negative by urinary GC-MS analysis.