Objectives The newborn screening (NBS) program in the Republic of Serbia has several decades of tradition, but it has not included any organic acidemias (OA). Therefore, this study aimed to establish the cut-offs of the corresponding NBS markers in the population of healthy newborns. Methods In dried blood samples (DBS) collected from 1,771 healthy newborns, we analyzed levels of propionylcarnitine (C3), isovalerylcarnitine (C5), and glutarylcarnitine (C5DC) using tandem mass spectrometry. Further we calculated the following ratios: C3/acetylcarnitine (C3/C2), C3/palmitoylcarnitine (C3/C16), C5/ free carnitine (C0), C5/C2, C5/C3, C5DC/octanoylcarnitine (C8), and C5DC/C0. Results The cut-offs for methylmalonic acidemia (MMA) or propionic acidemia (PA) were C3>5.73 μmol/L, C3/C2>0.23, and C3/C16>2.36. Based on the study findings, the screening results indicative for isovaleric acidemia (IVA) would include C5>0.372 μmol/L, C5/C0>0.020, C5/C2>0.019, and C5/C3>0.31. Finally, C5DC>0.303 μmol/L, C5DC/C8>7.1, and C5DC/C0>0.019 would justify further testing for glutaric acidemia type I (GA1). The cut-offs were satisfactorily validated via the comparison with worldwide estimates and data for several Caucasian populations. Conclusions The levels of the OA biomarkers in the Serbian population of healthy newborns have a distribution pattern similar to the other world populations. Therefore, the proposed cut-offs represent a reliable starting point for the future development of the OA NBS.