Capsaicin has been reported to exhibit an inhibitory effect on the P-glycoprotein (P-gp) function in vitro. To investigate its concentration-dependent effect in vivo, a sensitive assay that can characterize the absorption and disposition of capsaicin needs to be developed. This study reports the development of a sensitive LC-MS/MS assay for the determination of capsaicin in mouse plasma. The sample pretreatment involved a one-step extraction of 20 μL plasma with t-butyl methyl ether. Separations were achieved on a C18 column and the detection was performed on an LC-ESI-MS/MS by multiple reaction monitoring. The assay was linear over a wide concentration range from 0.325 to 650 ng mL−1 (r > 0.999), with a LLOQ of 0.325 ng mL−1. The developed method was applied to i.v. (dose 0.325 and 0.65 mg kg) and oral absorption (dose 40 mg kg) studies in mice. After i.v. injection, the t1/2,λz, Vz and CLs ranged from 0.13–0.16 h, 127.6–141.8 mL, and 547.3–775.4 mL/h, respectively. After oral administration, a secondary peak was observed and the terminal half-life was prolonged (1.51 h). Capsaicin was poorly absorbed, with the absolute oral bioavailability (F) ranging from 1.02% to 1.56%. The developed assay may be useful in studies where sample volumes are limited.