<p>Glycoconjugate vaccines rely on the accurate quantification of its active ingredient, also called conjugated saccharides (CS), which is usually estimated from the indirect measurement of total and free (unconjugated) saccharide to ensure product quality and efficacy. Conventional methods like HPAEC-PAD often require harsh and time-consuming sample pre-treatment, especially in multivalent formulations. In this study, we introduce a tetraplex sandwich immunoassay based on Luminex technology, capable of directly and simultaneously quantifying the conjugate saccharide in MenACWY vaccine without pre-treatments (e.g., hydrolysis, solid-phase extraction, and ultrafiltration steps). The new assay exhibits robust specificity, sensitivity, linearity, precision, and accuracy across a broad concentration range, and effectively discriminates conjugated saccharides from free saccharides and carrier protein. Stability studies under both accelerated and long-term conditions confirm the method’s suitability as a stability-indicating assay. This high-throughput, multiplex platform offers a robust and efficient alternative to traditional physico-chemical approaches, streamlining analytical workflows for increasingly complex glycoconjugate vaccines.</p> Graphical Abstract <p></p>