The reference methods used for sickle cell disease (SCD) screening usually include two analytical steps: a first tier for differentiating haemoglobin S (HbS) heterozygotes, HbS homozygotes and β-thalassemia from other samples, and a confirmatory second tier. Here, we evaluated a first-tier approach based on a fully automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform with automated sample processing, a laboratory information management system and NeoSicklesoftware for automatic data interpretation. A total of 6701 samples (with high proportions of phenotypes homozygous (FS) or heterozygous (FAS) for the inherited genes for sickle haemoglobin and samples from premature newborns) were screened. The NeoSickle software correctly classified 98.8% of the samples. This specific blood sample collection was enriched in qualified difficult samples (premature newborns, FAS samples, late and very late samples, etc.). In this study, the sensitivity of FS sample detection was found to be 100% on the Lille MS facility and 99% on the Dijon MS facility, and the specificity of FS sample detection was found to be 100% on both MS facilities. The MALDI-MS platform appears to be a robust solution for first-tier use to detect the HbS variant: it is reproducible and sensitive, it has the power to analyze 600–1000 samples per day and it can reduce the unit cost of testing thanks to maximal automation, minimal intervention by the medical team and good overall practicability. The MALDI-MS approach meets today’s criteria for the large-scale, cost-effective screening of newborns, children and adults.