MTH1 is a protein that is required for cancer cell survival and is overexpressed in cancer cells. TH588 and TH287 are two new compounds that inhibit the MTH1 protein. The inhibitors were tested in pharmacokinetic studies on mice. A bioanalytical method was developed and validated for determination in mice plasma. The method was based on protein precipitation followed by LC-MS/MS analysis. The separation was performed on an Ascentis Express RP-Amide C18 column. The mass spectrometer was operated in positive electrospray ionization mode and the analytes were determined with multiple reaction monitoring (MRM). Abundant monoisotopic fragments were used for quantification. Two additional fragments were used for conformational analysis. The recovery of the compounds in plasma varied between 61 and 91% and the matrix effects were low and ranged between -3% and +2%. The method showed to be selective, linear, accurate and precise, and applicable for preclinical pharmacokinetic studies of TH588 and TH287 in mouse plasma. Half-life (T1/2) was ≤3.5h and maximum concentration (Cmax) ranged between 0.82 and 338μM for the different administration routes and compounds.