A standard technique for performing purity measurements is UV absorbance with a spectrophotometer. Microvolume spectrophotometers are commonly used for the analysis of nucleic acid samples. They require a small sample volume (0.5\u20135.0 \u03bcl) and are economical, convenient and widely available. Typically, they measure concentration and purity readings for ssDNA, dsDNA and RNA and can provide meaningful insights into the quality of the sample.Due to some reported limitations with this method, we would recommend deploying Nanodrop UV spectrophotometry ONLY for purity ratio estimations (A260\/280 and A260\/230) of double-stranded or single-stranded DNA\/RNA, especially when destined for downstream applications such as DNA library preparation for whole-genome, amplicon and targeted sequencing.As an indicator of sample purity, the ratios of the absorbance values at 260 nm vs 280\u00a0nm (A260\/A280) and at 260 nm vs 230 nm (A260\/A230) need to be determined for each sample to ensure its suitability for downstream applications. The A260\/A280 provides insight into the type of nucleic acid present (dsDNA or RNA) as well as an indication of purity. Typically, protein contamination can be detected by a reduction in this ratio; RNA contamination can be detected by an increase in this ratio. In buffered solutions, pure dsDNA has an A260\/ A280 of 1.85\u20131.88. The A260\/A230 is a sensitive indicator of contaminants that absorb at 230 nm. These contaminants are significantly more numerous than those absorbing at 280 nm, and include chaotropic salts such as guanidine thiocyanate (GTC) and guanidine hydrochloride (GuHCl), EDTA, non-ionic detergents like Triton\u2122 X-100 and Tween\u00ae 20, proteins, and phenol. Substances like polysaccharides or free floating solid particles like silica fibers absorb at this wavelength, but will have a weaker effect. In buffered solutions, pure dsDNA has a higher A260\/A230 ratios at 2.3\u20132.4. A260\/A230 ratios typically produce a higher standard deviation than A260\/ A280 ratios and should be interpreted with care.This protocol has been adapted from nucleic acid purity measurement assays developed by ThermoFisher Scientific.This is an open-access protocol distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike.