The detection of thiols on extraterrestrial bodies could provide evidence for life, as well as a host of potential prebiological or abiological processes. Here, we report a novel protocol to analyze organic thiols by microchip CE with LIF detection. Thiols were labeled with Pacific Blue C5 maleimide and analyzed by MEKC. The separation buffer consisted of 15 mM tetraborate pH 9.2 and 25 mM SDS. The optimized method provided LODs ranging from 1.4 to 15 nM. The method was validated using samples collected from geothermal pools at Hot Creek Gorge, California, which were found to contain 2-propanethiol and 1-butanethiol in the nanomolar concentration range. These samples serve as chemical analogues to material potentially present in the reducing environment of primitive Earth and also at sulfurous regions of Mars. Hence, the protocol developed here enables highly sensitive thiol analysis in samples with complexity comparable to that expected in astrobiologically relevant extraterrestrial settings. This new protocol could be readily added to the existing suite of microfluidic chemical analyses developed for in situ planetary exploration; all that is required is the incorporation of two new reagents to the payload of an existing instrument concept.