Bacteria and yeast use secreted signal peptides, also known as pheromones, for cell-cell communication within their respective species. Recently, genetic modification has allowed for the extension and exploitation of this type of communication, to communication between organisms from different species and even from different kingdoms. This innovative approach is intended to allow for the large-scale production of specific compounds for applications in medicine and biotechnology while producing reduced amounts of by-products. Until now, the detection of signal peptides, which are often short-lived and only present in small amounts, is usually qualitative, non-selective, and time-consuming and/or requires the presence of additional cell types. Here, an ESI-LC-MS/MS method for the specific quantification of signal peptides from yeast (α- and P-factor) and bacteria (CSF) using a TSKgel column operating under HILIC conditions has been demonstrated. The influence of different matrices, their adsorption behavior, and their stability were investigated. In matrix, LOQs of 0.05 µM, 0.03 µM, and 0.02 µM were obtained for CSF, α-factor, and P-factor, respectively. Subsequently, the developed method was applied to the detection of yeast- and bacteria-specific peptides secreted by genetically modified yeasts. It could be demonstrated that under overexpressing conditions, α-factor and P-factor concentrations of 1 µM were measured, while for CSF concentrations as high as 2.5 µM was reached. Finally, the established method permits the simultaneous, quantitative detection of signal peptides in different matrices and without pre-concentration in near-real time, thus advancing the possibility of tracking cross-kingdom communication.