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Dynamic optical contrast imaging (DOCI) leverages a novel realization of temporally dependent measurements of tissue autofluorescence that allows the acquisition of specific tissue properties. A prospective series of patients with primary hyperparathyroidism was examined. Parathyroid lesions and surrounding tissues were collected; fluorescence decay images were acquired via DOCI. Ex vivo samples (81 patients) were processed for histologic assessment. DOCI extracts relative fluorescence decay information in a surgically relevant field of view with a clinically accessible acquisition time &lt;2\u00a0minutes. Analysis of DOCI revealed microscopic characterization sufficient for tissue type identification consistent with histology (<jats:italic>P<\/jats:italic> &lt;. 05). DOCI is capable of efficiently distinguishing parathyroid tissue from adjacent tissues. 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