This study compared ultrasound biomicroscopy with histopathology in discerning depth of eyelid lesions and tissue characteristics. Ultrasound biomicroscopy (50 MHz) was used to analyze lesion depth and tissue characteristics of eight patients with eyelid lesions referred to a university-based referral practice prior to excisional biopsy. This data was then compared to histopathologic findings. Lesion depth measured by ultrasound biomicroscopy was correlated with histopathologic depth with a linear regression. Ultrasound biomicroscopy correlated highly to histopathology with regards to tissue characteristics and lesion depth (R2 = 0.87). Ultrasound biomicroscopy accurately measures depth of eyelid lesions and accurately determines tissue characteristics.