While high-performance liquid chromatography (HPLC) is well-established for β-thalassaemia and haemoglobinopathies, phenotypic screening for α0-thalassaemia has been limited. To address this limitation, we aimed to translate the discovery of the α-thalassemia early eluting peak (αEEP) in HPLC into clinical practice by comparing its diagnostic performance with other existing methods (haemoglobin H inclusion test [HbHi] and immunochromatographic strip test [ICT]) in a multicentre setting, and elucidating the nature of the αEEP by liquid chromatography-tandem mass spectrometry (LC-MS/MS). With a cohort of 820 genotyped patients, the αEEP showed superior diagnostic performance in detecting --SEA (sensitivity 99.6%, specificity 100%) compared with HbHi (sensitivity 95.8%, P = 0.006; specificity 97.3%, P < 0.001) and ICT (sensitivity 95.8%, P = 0.006; specificity 75.4%, P < 0.001). Both HbHi and ICT showed reduced sensitivity in β-thalassaemia carriers versus non-carriers. ICT showed reduced specificity when Hb F ≥ 1% compared with < 1%. The αEEP remained robust across all subgroups. LC-MS/MS revealed a strong association between the αEEP and embryonic ζ-globin chains (P < 0.001). The αEEP offered cost reductions of 98.6% over HbHi and 97.3% over ICT. Collectively, the αEEP is a highly reliable and cost-effective marker for detecting --SEA carriers, enabling a novel "all-in-one" HPLC screening strategy for --SEA, β-thalassaemia and haemoglobinopathies. Trial registration number: not applicable.