Oligonucleotides including oligodeoxynucleotides (ODN) are usually quantified by measuring UV absorbance at 260 nm (OD260) in a separate step after HPLC purification or analysis. Once the value of OD260 of an oligonucleotide is obtained, its mass in micro grams or micro moles can be easily calculated based on its sequence. Such calculations are usually carried out using free online tools by simply imputing the sequence and the OD260 value. In this report, we describe a method to determine OD260 directly from the area of HPLC peak instead of obtaining the value in a separate step.