This protocol outlines the construction and operation of a DIY NanoDrop spectrophotometer for quantifying DNA at 260 nm - 280nm, utilizing a USB webcam, UV- LED, DVD diffraction grating, mirror, and blackbox enclosure. It includes step-by-step instructions for assembly, calibration, and measurement, tailored for use with Theremino Spectrometer software. Suitable for educational or low-budget laboratory settings.