Sphingosine-1-phosphate (S1P) acts as an extracellular signaling molecule with diverse biological functions. Tissues appear to have an S1P gradient, which is functionally relevant in the biological significance of S1P, although its existence has not been measured directly. Here, we report a highly sensitive method to determine the distribution of S1P, using a column-switching LC-MS/MS system combined with laser microdissection (LMD). Column switching using narrow core Capcell Pak C18 analytical and trap columns with 0.3 mm inner diameter improved the performance of the LC-MS/MS system. The calibration curve of S1P showed good linearity (r>0.999) over the range of 0.05–10 nM (1–200 fmol/injection). The accuracy of the method was confirmed by measuring S1P-spiked laser microdissected mice tissue sections. To evaluate our S1P analytical method, we quantified S1P extracted from micro-dissected mouse brain and spleen. These results show that this method can measure low S1P concentrations and determine S1P distribution in tissue microenvironments.