Oligodeoxynucleotides (ODNs) synthesized on an automated synthesizer are usually purified using reversed-phase (RP) or ion-exchange HPLC. Their purity is also usually determined using HPLC. For both preparative and analytical HPLC, the elution profile is mostly generated using a UV-Vis detector with the wavelength set to 260 nm. Quantities of ODNs are typically documented using optical density at 260 nm, which is abbreviated as OD260. It is defined as the UV absorbance at 260 nm (A260) of the ODN to be quantified dissolved in 1 mL of water with a light path of 1 centimetre. The value of OD260 is usually determined separately after an ODN is purified by HPLC using an UV spectrometer or other UV based apparatus such as a NanoDrop. Because the peak area in an HPLC profile is a quantitative measure of the UV absorbance of the ODN eluted within the peak, we reasoned that a separate step for the determination of OD260 using a UV spectrometer as we usually do is unnecessary, and the OD260 value can be determined directly using the peak area in the HPLC profile. Here, we describe the establishment of a correlation graph between HPLC peak areas and OD260 values and demonstrate its use for the determination of OD260 using HPLC peak area without having to measure UV absorbance separately.