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The lipid layer of the tear film limits evaporation during the interblink interval and also affects tear stability. This study was designed to validate a new software application designed to characterize the tear film lipid layer through texture and color pattern recognition.Using the Tearscope-plus (slit-lamp magnification ×200), the lipid layer was examined in 105 healthy young adults, and interference photographs were acquired with a Topcon DV-3 digital camera. The photographs were classified by a clinician experienced in examining lipid layer patterns (LLPs), and these classifications were used as the reference standard (reference examiner). Next, LLPs were graded using the new software and further by 2 observers (observer 1 and observer 2) with experience in examining the ocular surface.Strong correlation was detected between the categories determined by the new application and reference examiner (Cramer V 0.85–0.93, P < 0.001). The classifications made using the new application and by observer 1 and observer 2 were also consistent although correlation was weaker (Cramer V 0.56–0.87, P < 0.001). For thinner LLPs, greatest correspondence with the reference was observed using the new software (96.6%), whereas the 2 observers showed better agreement when grading thicker patterns. Notwithstanding, agreement between the 2 observers and the reference examiner was good with at least 81% matched classifications. Best agreement (96.2%) was noted between the new method and observers 1 and 2 for recognizing meshwork patterns, whereas observers 1 and 2 showed greatest correspondence when classifying color fringe patterns.The new application can objectively categorize LLPs using the Tearscope-plus.