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Monitoring for mycotoxins in food or feed matrices is necessary to ensure the safety and security of global food systems. Due to a lack of standardized methods and individual laboratory priorities, most institutions have developed their own methods for mycotoxin determinations. Given the diversity of mycotoxin chemical structures and physicochemical properties, searching databases, and comparing data between institutions is complicated. We previously introduced incorporating a retention index (RI) system into liquid chromatography mass spectrometry (LC-MS) based mycotoxin determinations. To validate this concept, we designed an interlaboratory study where each participating laboratory was sent N-alkylpyridinium-3-sulfonates (NAPS) RI standards, and 36 mycotoxin standards for analysis using their pre-optimized LC-MS methods. Data from 44 analytical methods were submitted from 24 laboratories representing various manufacturer platforms, LC columns, and mobile phase compositions. Mycotoxin retention times (tR) were converted to RI values based on their elution relative to the NAPS standards. Trichothecenes (deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol) showed tR consistency (+/- 20-50 RI units, 1-5 % median RI) regardless of mobile phase or type of chromatography column in this study. For the remaining mycotoxins tested, the RI values were strongly impacted by the mobile phase composition and column chemistry. The ability to predict tR was evaluated based on the median RI mycotoxin values and the NAPS tR. These values were corrected using Tanimoto coefficients to investigate whether structurally similar compounds could be used as anchors to further improve accuracy. This study demonstrated the power of employing an RI system for mycotoxin determinations, further enhancing the confidence of identifications. Monitoring for mycotoxins in food or feed matrices is necessary to ensure the safety and security of global food systems. Due to a lack of standardized methods and individual laboratory priorities, most institutions have developed their own methods for mycotoxin determinations. Given the diversity of mycotoxin chemical structures and physicochemical properties, searching databases, and comparing data between institutions is complicated. We previously introduced incorporating a retention index (RI) system into liquid chromatography mass spectrometry (LC-MS) based mycotoxin determinations. To validate this concept, we designed an interlaboratory study where each participating laboratory was sent N-alkylpyridinium-3-sulfonates (NAPS) RI standards, and 36 mycotoxin standards for analysis using their pre-optimized LC-MS methods. Data from 44 analytical methods were submitted from 24 laboratories representing various manufacturer platforms, LC columns, and mobile phase compositions. Mycotoxin retention times (tR) were converted to RI values based on their elution relative to the NAPS standards. Trichothecenes (deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol) showed tR consistency (± 20-50 RI units, 1-5 % median RI) regardless of mobile phase or type of chromatography column in this study. For the remaining mycotoxins tested, the RI values were strongly impacted by the mobile phase composition and column chemistry. The ability to predict tR was evaluated based on the median RI mycotoxin values and the NAPS tR. These values were corrected using Tanimoto coefficients to investigate whether structurally similar compounds could be used as anchors to further improve accuracy. This study demonstrated the power of employing an RI system for mycotoxin determinations, further enhancing the confidence of identifications.Copyright © 2025. Published by Elsevier B.V.