This research examines how and in what sense the development of multiplex detec-tion platform of genitourinary infectious pathogens based on mass spectrometry is poten-tially advantageous in terms of diagnosis. Within the process of examination, multiplex PCR, nicking reaction, and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis were applied to enhance the detection efficiency through multiplex system. A condition for multiplex PCR method was settled in order to amplify all 13 types of genes from selected pathogens and $\beta$ -globin house-keeping gene. The multiplex PCR in this strategy enabled detection of samples of not only single infection but also multiple infections, which is very common case in infected patients. In order to ap-ply MALDI-TOF MS analysis to amplified PCR products, nicking reaction method was established in order to generate short DNA fragments that are used as mass markers. The mass markers used in this strategy are designed to be target-specific in order to enable the analysis of mass spectrometry to be performed in a multiplex manner as well. With this de-tection strategy, both single- and multiple-infected clinical human samples of 13 target pathogens were successfully diagnosed. The results show that this novel detection strategy applying mass spectrometry has a potential to be a convenient tool for diagnosis of Sexually Transmitted Diseases (STDs).