Solasodine, a major ingredient in Solanaceae family, has various biological functions such as inducing neurogenesis, anticonvulsant and anti-tumor. Its risk assessment has also drawn public attention. However, little is known about its oral bioavailability and metabolic process. In this study, an liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of solasodine in mice dried blood spot (DBS) samples. To block nonspecific adsorption, DBS samples were pretreated with bovine serum albumin (BSA) and then extracted with ethyl acetate. This method was applied to a pharmacokinetic and bioavailability study of solasodine. The absolute bioavailability was only 1.28%. Thereafter, its metabolites in mice were characterized using an ultra-performance liquid chromatography Q-Exactive high-resolution mass spectrometer (UHPLC-QE-HRMS). Several isomeric metabolites were well separated and differentiated using their retention time, fragmentation pathways and correspondingly fragmentation rules of solasodine. As a result, 21 metabolites were characterized including 16 phase I and 5 phase II metabolites. The proposed metabolic pathways showed that solasodine mainly experienced oxidation, dehydration, dehydrogenation and sulfation. These results could help us to better understand the efficacy and safety of solasodine. © 2021 Elsevier B.V.