We describe an HPLC analytical technique for detection of minute amounts of oil-based Red 26 dye used for marking insects. Chromatographic separation was achieved by an isocratic elution on an Inertsil 100A, 5µ silica column using hexane: isopropyl alcohol (98:2) as the mobile phase and with absorption detection at 520 nm. This newly developed technique is fast and very sensitive, with a detection threshold at 0.1 ng and quantitation down to 0.3 ng per pink bollworm adult. Using the newly developed technique we were able to correlate the titer of dye extracted from adult moths to the amount of dye included in the larval diet. This method is currently accepted by the U.S. cotton industry as a preferred technique for distinguishing artificial diet-reared sterile from wild pink bollworm moths.