Methanethiol (MeSH) is a pivotal volatile sulfur compound in plant, human, and environmental chemistry, but its study is hindered by analytical challenges. Here, we present an accessible fluorometric two-component assay toolkit for quantitative assessment of its content and enzymatic degradation by methanethiol oxidase (MTO). The first assay sensitively detects MeSH with a limit of detection of 124 nM using a novel “tube-in-tube” design for efficient MeSH capture, circumventing the need for complex chromatography and expensive instrumentation. The second assay is a real-time kinetic MTO activity assay. Using this toolkit, we (i) established an inverse correlation between MTO activity and MeSH levels in a colon cancer cell line, (ii) quantified MeSH as a key scent marker in various types of cheese, (iii) evaluated MeSH release from the selected Brassica and Allium species and (iv) determined its production in aquatic systems. The MeSH metabolism toolkit presented here will provide researchers with simple, sensitive and specific assays to elucidate multifaceted roles of MeSH and MTO in human pathophysiology and to accelerate discoveries in a broad spectrum of scientific disciplines.