The Autism Diagnostic Observation Schedule, Second Edition (ADOS-2) has been regarded as the gold standard assessment of autism spectrum disorder (ASD). While clinical validity of ADOS-2 Modules 1-4 have been extensively studied, there has been very limited research examining the clinical validity of ADOS-2 Toddler Module. The goal of this study was to examine alignment of the ADOS-2 Toddler Module classification with clinicians' determination of ASD, with assessing cut-off scores for diagnosing ASD in toddlers. A total of 412 toddlers ages 12-30 months who received ADOS-2 Toddler Module as well as a best estimate clinical (BEC) diagnosis, were included in this study. ADOS-2 Toddler Module cut-off scores were determined using the appropriate algorithms (Algorithm 1 for toddlers aged 12-20 months and those aged 21-30 months with <5 words, Algorithm 2 for toddlers aged 21-30 months with 5 words or more). Receiver operating characteristic (ROC) curves were used to assess cut-off scores that optimized sensitivity and specificity when compared against BEC diagnosis. The selected cut-off scores were examined using frequency tables to compare ADOS-2 classification against BEC diagnosis. For Algorithm 1, classification statistics were optimized at the cut-off score of 12 with an area under the curve (AUC) of 0.92. For Algorithm 2, classification statistics were optimized at the cut-off score of 10 with an AUC of 0.96. The ADOS-2 Toddler Module classification is strongly aligned with BEC diagnosis. The optimal cut-off scores identified in the current study reflect the same results configured by the prior study. LAY SUMMARY: ADOS-2 Toddler Module has been widely used for the ASD assessment, but there have been limited research on its clinical validity. This study is the first replication of the ADOS-2 Toddler Module with a large independent sample. We examined alignment of the ADOS-2 Toddler Module classification with clinicians' determination of ASD, with assessing cut-off scores, and confirmed the clinical validity of ADOS-2 Toddler Module. Cut-off scores of ADOS-2 Toddler Module cited in the manual yielded best clinical utility for diagnosing ASD in toddlers.