Serological diagnosis during neonatal screening is crucial in disease prevention. Among the infectious diseases, the most common are toxoplasmosis, rubella, and cytomegalovirus. Traditional diagnostic methods are used to detect a single infectious agent per test. The use of multiplex detection methods increases productivity and reduces the amount of material used, resulting in a more efficient test from a technical, environmental, and economic point of view. The study’s objective was to evaluate the performance of a new diagnostic method aimed at neonatal screening using the multiplex platform of magnetic microspheres from the company Luminex Corporation. For this, tests were carried out for analytical validation of the diagnostic product developed following the rules of the National Health Surveillance Agency (ANVISA) of Brazil. The parameters evaluated were repeatability, reproducibility, linearity, robustness, high dose, minimum detection limit, and analytical specificity. All data obtained met the acceptance criteria of RDC 166/17 of 2017 for the use of the diagnostic product in the national territory. Repeatability and reproducibility tests showed a CV of less than 15% between replicates of the same operator and different operators. The kit showed linearity throughout the operating range with R2 above 0.990, and no effect of high-efficiency dose was observed in the chosen working dilution. In addition, the kit did not show interference from the matrix with the results, and it was observed that small and deliberate changes in the incubation time of each reagent did not have a significant effect on the data obtained.