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A state of limbal stem cell deficiency (LSCD) can be secondary to a number of etiologies, resulting in either a reduction in the total number of limbal stem cells or an abnormality in stem cell function. Initially, the epithelium becomes irregular and hazy; however, this condition may progress to persistent corneal epithelial defects, stromal scarring, ulceration, and even perforation. Since LSCD secondary to a variety of etiologies may be reversible, and various factors are prognostic of disease progression, timely diagnosis is important. This review will describe current knowledge of diagnostic techniques for LSCD and understanding of epithelial stem cell function.Conjunctivalization, regarded as the most reliable clinical finding diagnostically, can be identified as late staining of epithelium with fluorescein. While identifying loss of the palisades of Vogt by slit-lamp examination, can provide a high suspicion of LSCD, but this is not diagnostic. Impression cytology is a simple, noninvasive technique that aids in the diagnosis of LSCD, but a negative result also cannot rule out the diagnosis. Recent findings have also shown that imaging techniques including in-vivo confocal microscope and optical coherent tomography can also aid in diagnosing LSCD; however, several challenges remain before these techniques become standard diagnostic methods in clinical practice. Meanwhile, determination of the absence of limbal epithelial crypts and focal stromal projections using image reconstruction techniques may assist in the diagnosis of LSCD. Furthermore, histologic markers may help not only to improve sensitivity and specificity of conventional techniques in diagnosis of LSCD, but also to identify human limbal stem cells and determine their number and function in LSCD.Efforts to develop and improve techniques for diagnosing LSCD are ongoing. Increased knowledge of limbal stem cells and components of their niches may not only help in understanding the pathogenesis of LSCD but may improve its diagnosis, thereby ameliorating the prognosis of patients with this devastating disease.