Thymosin (4) (T-4) is a peptide present in almost any tissue and in extracellular media in mammals, having multiple amazing functions as wound healing, stimulation of angiogenesis, and suppression of inflammation. This study describes its determination in saliva through CE-MS using multiple ions monitoring scan mode by isolating the four most intense multicharged ions present in the MS spectra of the peptide. This scan modality, by reducing the baseline noise and interferences, increases the sensitivity and specificity in biological matrices. The CE-MS separation was optimized by studying different parameters influencing CE analysis, sample injection, and MS ionization, that is, the nebulizer gas flow, the sheath liquid, and BGE composition. The proposed technique can unambiguously identify in short time T-4 in saliva after a very fast and reduced sample pretreatment procedure. The method was validated for quantitation showing linearity of the response in the range 0.25 (lower limit of quantification) to 4 M (average R-2 0.996 +/- 0.005) and intra- and interassay precision and accuracy at three different concentrations with RSD values in the range of 7-16%. It was successfully applied to the analysis of T-4 in whole saliva showing a variable peptide content from individual to individual (in the range of 0.3-1.4 M) and in different days from the same individual. CE-MS in multiple ions monitoring scan mode provides a fast, selective, and economic method requiring only very few microliters of sample.