Diacetylmonoxime is widely used in analytical chemistry as a reagent for the determination of urea and its derivatives. It reacts with urea in the presence of acid and heat to form a colored complex, which can be measured spectrophotometrically, making it valuable in clinical diagnostics for assessing kidney function and monitoring urea levels in blood and urine.

In biochemistry, it serves as a chelating agent for metal ions, particularly in studies involving enzyme inhibition and metal ion interactions. Its ability to form stable complexes with metals makes it useful in research focused on understanding metal-dependent enzymatic processes.

Additionally, it is employed in organic synthesis as a building block for the preparation of various heterocyclic compounds. Its reactivity with carbonyl groups and amines facilitates the creation of complex molecular structures, which are important in pharmaceutical and agrochemical development.

In industrial applications, diacetylmonoxime is used in the production of certain polymers and resins, where it acts as a cross-linking agent or stabilizer, enhancing the material properties of the final product.