Being traditionally utilized mainly as appetizers and herbal teas as well as used to ease abdominal pains, colds and gastrointestinal issues, the genus Salvia L. has gained significant consideration owing to its remarkable phytochemicals and industrial importance. The present study aimed to develop and validate an LC-MS/MS method for the qualitative and quantitative investigation of 19 fingerprint phytochemicals in six endemic Salvia species. The validation parameters of the developed LC-MS/MS method were repeatability (intermediate precision), recovery (accuracy), limits of detection and quantification, linearity and uncertainty (U% at 95% confidence level (k = 2)). Reversed-phase HPLC separation and mass spectrometry parameters were optimized for each analyte. Ethanol extracts of the studied Salvia species collected in three consecutive years were screened for their fingerprint phytochemicals by using the developed and validated LC-MS/MS method. Moreover, studied Salvia species were subjected to multivariate analysis such as principal component analysis techniques to demonstrate the variabilities in phytochemical contents by years and parts of the samples. Roots, flowers, leaves, branches and whole plant of the Salvia species collected in 2015, 2016 and 2017 were used for the analyses. It was observed that the roots and branches of Salvia species were similar in terms of their salvianolic acid A, caffeic acid, and 6,7-dehydroroyleanone components. Accordingly, apigenin, rosmarinic acid, luteolin 7-O-glucoside, caffeic acid, salvianolic acid B, and 6,7-dehydroroyleanone were notable phytochemicals that were present in the studied Salvia species.