Noninvasive localized measurement of extracellular pH in cancer tissues can have a significant impact on the management of cancer. Despite its significance, there are limited approaches for rapid and noninvasive measurement of local pH in a clinical environment. In this study, we demonstrate the potential of noninvasive topical delivery of Alexa-647 labeled pHLIP (pH responsive peptide conjugated with Alexa Fluor® 647) to image changes in extracellular pH associated with head and neck squamous cell carcinoma using widefield and high resolution imaging. We report a series of preclinical analyses to evaluate the optical contrast achieved after topical delivery of Alexa-647 labeled pHLIP in intact fresh human tissue specimens using widefield and high-resolution fluorescence imaging. Using topical delivery, Alexa-647 labeled pHLIP can be rapidly delivered throughout the epithelium of intact tissues with a depth exceeding 700 µm. Following labeling with Alexa-647 labeled pHLIP, the mean fluorescent contrast increased four to eight fold higher in clinically abnormal tissues as compared to paired clinically normal biopsies. Furthermore, the imaging approach showed significant differences in fluorescence contrast between the cancer and the normal biopsies across diverse patients and different anatomical sites (unpaired comparison). The fluorescence contrast differences between clinically abnormal and normal tissues were in agreement with the pathologic evaluation. Topical application of fluorescently labeled pHLIP can detect and differentiate normal from cancerous tissues using both widefield and high resolution imaging. This technology will provide an effective tool to assess tumor margins during surgery and improve detection and prognosis of head and neck cancer.