Herein, we describe a highly efficient, non-complicated, and non-organic procedure to overcome the negative effect of chemotherapeutic drugs on the quality of the extracted DNA by applying several modulations in cell washing, suspension, and lysis of cells treated with these drugs. In this protocol, 500µl were extracted from patients who received systemic sessions of chemotherapy. The validity of this protocol for digestion with restriction endonucleases and both conventional and real-time polymerase chain reaction were tested. This protocol proved obvious purity (1.8±0.02 and 2.1±1.2, for A_260/280 and A_260/230, respectively) and adequate yields (10±2.24) µg/100 ml. The positive results of validation experiments proved the validity of the extracted DNA for downstream applications of molecular biology. In addition to the proven efficiency of this protocol to extract DNA from normal samples, its validity was also confirmed from patients who were exposed to chemotherapy. This entails a novel approach to extract a molecular biology grade DNA without having inhibitors against enzymes used in digestion, amplification, and subsequent sequencing even after the systemic sessions with several doses of chemotherapy.