Noninvasive optical imaging technology has the potential to improve the accuracy of disease detection and predict treatment response. Pathology provides the critical link between the biological basis of an image or spectral signature and clinical outcomes obtained through optical imaging. The validation of optical images and spectra requires both morphologic diagnosis from histopathology and parametric analysis of tissue features above and beyond the declared pathologic "diagnosis." Enhancement of optical imaging modalities with exogenously applied biomarkers also requires validation of the biological basis for molecular contrast. For an optical diagnostic or prognostic technology to be useful, it must be clinically important, independently informative, and of demonstrated beneficial value to patient care. Its usage must be standardized with regard to methods, interpretation, reproducibility, and reporting, in which the pathologist plays a key role. By providing insight into disease pathobiology, interpretive or quantitative analysis of tissue material, and expertise in molecular diagnosis, the pathologist should be an integral part of any team that is validating novel optical imaging modalities. This review will consider (1) the selection of validation biomarkers; (2) standardization in tissue processing, diagnosis, reporting, and quantitative analysis; (3) the role of the pathologist in study design; and (4) reference standards, controls, and interobserver variability.