Rare diseases have a low prevalence in the population, they occur in less than five individuals per 10 000 people and are mostly diagnosed late. Inherited metabolic diseases are rare diseases that occur as a result of reduced activity of enzymes, transporters or cofactors. NBS is a procedure for the organized and systematic detection of congenital diseases in the newborn population with the purpose of their timely recognition. In the Republic of Croatia NBS started in 1978, and today screening is performed for nine inherited metabolic diseases. Screening tests are not diagnostic tests, they indicate newborns who may have a certain metabolic disorder and exclude those who probably do not have these disorders. Laboratories that perform NBS for classic homocystinuria and vitamin B12 deficiency use different procedures. Carrying out an extended NBS using the LC-MS/MS method from a DBS sample implies the analysis of amino acids and acylcarnitine, as primary markers, by derivatized or non-derivatized methods. Due to the large number of false positive results after the analysis of the primary markers C3, C2, Met and Phe for homocystinuria and vitamin B12 deficiency, it is necessary to include more specific second-tier tests (tHcy, MMA) in the procedure. The aim of this paper is to prepare the extension of newborn screening in Croatia for the mentioned diseases. For this purpose, a comparison was made of the six-month results of measuring the analytes of interest (C3, C2, Met and Phe) and their mutual ratios in the NBS of the Croatia with the values of the same analytes used in the procedures of the newborn programs of Austria, Italy, Germany and Spain. It was estimated that by applying the criteria from the procedures of the mentioned countries to the results of the NBS of the Republic of Croatia, for a six-month period, the number of samples for measuring Hcy in the second-tier test would be acceptable for conducting the NBS for homocystinuria and vitamin B12 deficiency in Croatia.