The present invention relates to a method of directly detecting, using a mass-spectrometry method, whether a microorganism contained in a sample is resistant to antibiotics, and a kit for detection used therewith. More particularly, the present invention relates to a method and kit for directly detecting an antibiotic hydrolase secreted by a microorganism resistant to antibiotics, thereby directly determining whether the microorganism is resistant to antibiotics. According to the present invention, it is possible to very simply and immediately confirm whether a specific strain is resistant to antibiotics in the field. In particular, a complicated pretreatment process such as proteolysis is not performed, and a complicated identification process of calibrating and then combining the obtained results is not performed. Accordingly, it is possible to realize a method of easily confirming whether antibiotic resistance occurs in just a dozen minutes, compared to a conventional technology in which it takes several days to confirm whether antibiotic resistance occurs, and a simple diagnostic kit used therewith.