Background: Detection of microsatellite instability (MSI) is currently recommended for all patients with colorectal cancer (CRC). Current clinical reference methods are immunohistochemical staining of mismatch repair proteins and/or PCR analysis of frequently mutated short tandem repeat regions of DNA. The Idylla™ MSI Test prototype is being developed using a new set of short homopolymers capable of faster detection with greater specificity and selectivity compared to current methods. Methods: Prototype Idylla™ MSI Test cartridges were developed up to a finalized design. Repeat length of a novel set of 7 biomarkers was determined on 348 formalin-fixed and paraffin-embedded (FFPE) CRC samples using these prototype tests which allow a complete automated workflow including sample preparation, DNA amplification followed by melting curve analysis and automated interpretation. Patient samples of several clinical sites and different ethnic groups were included to assess robustness of marker selection. All samples were additionally screened with a reference methodology for MSI detection (Promega MSI analysis system). Results: One hundred twenty-seven (36.5%) and 116 (33.3%) samples were classified as MSI-high (MSI-H), and 209 (60.1%) and 220 (57.3%) samples were classified as microsatellite stable (MSS) by Idylla™ and Promega respectively, while 12 samples (3.4%) could not be classified by either methodology. Concordance analysis revealed an overall agreement of 96.1% (93.4%-97.7% 95% CI). Fourteen cases were MSI-H by Idylla™ but MSS (11) or invalid (3) by Promega; with a median of 3/7 positive markers on Idylla™. Conclusions: This study validated the novel MSI biomarkers to discriminate MSI-H from MSS status on a large and diverse set of CRC samples. It also demonstrated the possibility of a fully automated analysis for MSI testing. The prototype Idylla™ MSI Test is compatible with the fully integrated Idylla™ platform providing accurate and reliable results within 150 minutes from just one FFPE tumour section (no reference sample required).