A method was developed to quantify neurotensin (NT) fragment [8–13] and a novel NT[8–13] derivative, KK1, in human and rat serum utilizing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The method allows for simultaneous quantification of the major NT[8–13] metabolite, NT[9–13] (according to molecular mass), and detection of the major KK1 metabolite, KK1M (according to molecular mass). The degradation rates of NT[8–13] and KK1 were calculated to be 24.1±1.0 and 193±8min in human serum and 5.90±0.22 and 153±4min in rat serum, respectively. The method utilizes a novel sample drying technique and spectrum acquisition protocol. In addition, an internal standard dissimilar in structure to the analytes was used. This method may be broadly applicable to the quantification of NT[8–13] and other peptide analogues of varying structure.