Face perception and emotion categorization are widely investigated under laboratory conditions that are devoid of real social interaction. Using mobile eye-tracking glasses in a standardized diagnostic setting while applying the Autism Diagnostic Observation Schedule (ADOS-2), we had the opportunity to record gaze behavior of children and adolescents with and without Autism Spectrum Conditions (ASCs) during social interaction. The objective was to investigate differences in eye-gaze behavior between three groups of children and adolescents either (1) with ASC or (2) with unconfirmed (...) diagnosis of ASC or (3) with neurotypical development (NTD) during social interaction with an adult interviewer in a diagnostic standard situation using the ADOS-2. In a case control study, we used mobile eye-tracking glasses in an ecologically valid and highly standardized diagnostic interview to investigate suspected cases of ASC. After completion of the ASC diagnostic gold standard including the ADOS-2, the participants were assigned to two groups based on their diagnosis (ASC vs. non-ASC) and compared with a matched group of neurotypically developed controls. The primary outcome measure is the percentage of total dwell times assessed for different areas of interest (AOI) with regard to the face and body of a diagnostic interviewer and the surrounding space. Overall, 65 children and adolescents within an age range of 8.3–17.9 years were included in the study. The data revealed significant group differences, especially in the central-face area. Previous investigations under laboratory conditions gave preferential attention to the eye region during face perception to describe differences between ASC and NTD. In this study – using an ecologically valid setting within a standard diagnostic procedure – the results indicate that neurotypically developed controls seem to process faces and facial expressions in a holistic manner originating from the central-face region. Conversely, participants on the Autism Spectrum (tAS) seem to avoid the central-face region and show unsystematic gaze behavior, not using the preferred landing position in the central-face region as the Archimedean point of face perception. This study uses a new approach, and it will be important to replicate these preliminary findings in future research. (shrink)