<p>A simple, cost-effective, metal-free, and high-yielding protocol has been developed for the synthesis of hydroxamic acid derivatives from N-α-protected amino acids and aryl carboxylic acids using an inexpensive reagent, methanesulfonic anhydride. In this method, N-α-protected amino acids and aryl carboxylic acids are treated with methanesulfonic anhydride in the presence of Hünig’s base (DIPEA) to generate the corresponding mixed anhydride intermediates, which are subsequently reacted with hydroxylamine via a telescopic process to afford the desired hydroxamic acid derivatives. This approach offers several advantages, including the use of readily available and economical reagents, high product yields, facile removal of by-products such as methanesulfonic acid, unreacted starting materials, and DIPEA, and the generation of products with high purity. Notably, the protocol is compatible with various protecting groups, including Boc, Fmoc, and Cbz. The efficiency and generality of the optimized procedure were demonstrated through its application to a wide range of protected amino acids and aryl carboxylic acids, consistently yielding excellent results. Furthermore, the biological potential of the synthesized hydroxamic acid derivatives was evaluated through in silico ADME profiling and molecular docking studies against <i>E. coli</i> DNA gyrase B and CYP51. These investigations enabled the assessment of key interactions between the synthesized compounds and enzyme active sites. Docking results revealed that several hydroxamic acid derivatives exhibited stronger binding affinities than the reference standards, indicating their potential for further in vitro biological evaluation.</p> Graphical Abstract <p></p>