ObjectivesThe purpose of the study was to determine interobserver and intraobserver agreement, repeatability, and intrasubject variation of the detection of Demodex infestation in eyelids of blepharitis patients using in vivo confocal microscopy (IVCM). MethodsEighty-three eyes of 42 blepharitis patients were included in the study. All eyelids were evaluated from temporal to nasal with IVCM using section mode and 10 lashes with their follicles were imagined, and every image with suspicion of Demodex infestation was recorded. Two experienced and two inexperienced ophthalmologists were masked for the diagnosis and interpreted the IVCM images regarding the presence of Demodex infestation with a 3-week interval. Interobserver and intraobserver agreements were calculated with Cohen's kappa and its variant statistics between and within experienced observers and between inexperienced observers. ResultsWhile average sensitivity for the diagnosis of demodicosis in IVCM images was 83.35% for experienced and 51.35% for inexperienced observers, the average positive predictive value was 88.6% for experienced observers and 91.05% for inexperienced ones. Interobserver agreement between experienced observers was moderate (κ = 0.529) and intraobserver agreements within experienced observers were perfect (κ = 0.918 for observer-1; κ = 0.958 for observer-2). Interobserver agreement between inexperienced observers was poor (κ = 0.162) and intraobserver agreements within inexperienced observers were fair (κ = 0.427 for observer-3; κ = 0.475 for observer-4). ConclusionThe sensitivity, interobserver and intraobserver agreements in IVCM image analysis for the detection of Demodex infestation were highly associated with the clinical experience on IVCM imaging. In the hands of an experienced clinician, IVCM could be reliable for the diagnosis of ocular demodicosis.