Epidermal lipid barrier is composed by ceramides, cholesterol and free fat acids that maintain stability and protection. Fat-soluble dyes like Sudan and Oil Red are used for assessment of cell lipids, which is important for the detection of problems associated to atopic dermatitis. This study aimed to standardize and validate a technique for lipids assessment in normal dog epidermis using specific histological dyes, computerized image evaluation and observation of different pathologists. For standardization purposes, staining technique was developed and semiquantitative visual scale estimation and digital image processing technique were accomplished in 25 skin samples from healthy dogs. For validation purposes, the performance of the computerized evaluation was tested in 10 atopic and 20 healthy dogs. Skin samples were obtained from axillary and dorsal-cervical areas, processed by frozen sections and stained with either Oil Red or Sudan III. Images were analyzed by ImageJ® freeware by color deconvolution method. The amount of lipids in the epidermis was represented by positively stained epidermic area. Histological evaluations by two pathologists (- to ++++) were considered to validate the exams. Labeling of epidermal lipids was efficient in both staining techniques. Data presented by ImageJ® are based on width, intensity and area of the selected image. Values attributed to those data demonstrated that Sudan III represents better what is seen by pathologists. Sudan III dye presented width and area values equivalent to those observed by pathologists, while Oil Red presented only compatible width. Epidermal lipid assessment by computerized analysis was proven efficient and specific.