Diacetylmonoxime is widely used in analytical chemistry for the determination of urea in biological samples. It reacts with urea in the presence of an acid and heat to form a colored complex, which can be measured spectrophotometrically. This method is particularly useful in clinical laboratories for assessing kidney function and diagnosing renal disorders.

In the field of agriculture, it serves as a reagent for detecting and quantifying urea in soil and water samples, aiding in the assessment of nitrogen levels and the effectiveness of fertilizers. Additionally, it is employed in the synthesis of various organic compounds, including pharmaceuticals and agrochemicals, due to its ability to act as a versatile intermediate. Its applications also extend to research laboratories, where it is used in studies involving enzyme activity and nitrogen metabolism.