Abstract <p>Dialdehydes are predominantly used as disinfectants, as these compounds are characterized by a broad spectrum of antimicrobial activity. Of monoaldehydes, formaldehyde is used to a limited extent because of its higher toxicity. This paper describes the conditions for the simultaneous derivatization of formaldehyde, glyoxal, glutaric aldehyde, and orthophthalic aldehyde with 2,4-dinitrophenylhydrazine. The reaction is carried out in an acetonitrile−methanol mixture at 50°C in an ultrasonic bath using trifluoroacetic acid as a catalyst. The best separation of the mixture components is achieved on a C18 column in a gradient elution mode with acetonitrile and an acetate buffer solution (pH 5.4) at variable flow rates. The linearity range for formaldehyde is 2.51 to 20.0 mg/L, for glutaraldehyde 4.92 to 21.9 mg/L, for orthophthalaldehyde 1.98 to 6.94 mg/L, and for glyoxal 2.00 to 10.0 mg/L. The limits of detection for formaldehyde, glyoxal, glutaraldehyde, and orthophthalaldehyde are 0.453, 0.177, 0.967, and 0.760 mg/L, respectively. The developed procedure was successfully applied to the simultaneous determination of aldehydes in disinfectants.</p>