<p>Aldehydes have been primarily associated with certain types of cancer, making them potential biomarkers. This work focuses on the development of a sensitive extraction-derivatization methodology for the analysis of six aldehydes: pentanal, hexanal, heptanal, octanal, nonanal, and decanal in urine samples from control, smokers, and lung cancer patients as well as its possibility and prospects in blood plasma samples. Octyl-functionalized silica gel particles were used to extract aldehydes from the biological matrix, followed by derivatization of the analytes with 1-naphthalenyl hydrazine and analysis by HPLC-FLD. The methodology was optimized using a Plackett-Burman and central composite designs. Under optimal conditions found, calibration lines were constructed, obtaining limit of detections (LODs) in the interval of 0.55–0.80&#xa0;µg L⁻¹. This methodology was subsequently validated and applied to the determination of aldehydes in urine samples. The proposed derivatization methodology is efficient, with adequate precision (expressed as relative standard deviation &lt; 10%) and accuracy (90.97–110.03%). This methodology exhibits high sensitivity, precision, and accuracy, making it ideal for the determination of aldehydes in urine samples, which can be extrapolated to other biological samples such as blood plasma samples.</p> Graphical abstract <p></p>