We present a novel analytical platform for peptides quantitative assays in biological matrices based on microscale liquid chromatography fractionation and matrix-assisted laser desorption/ionization mass spectrometric detection using the selected reaction monitoring (SRM) mode. The MALDI source was equipped with a high frequency Nd:YAG laser (1000 Hz) and mounted on a triple quadrupole/linear ion trap mass spectrometer (MALDI-QqQ(LIT)). Compared to conventional LC-ESI-SRM/MS, the separated analytes are "time-frozen" onto the MALDI plate in fractions, and navigation through the LC chromatogram makes it possible to perform SRM experiments as well as enhanced product ion spectra acquisition for confirmatory analyses without time constraints. The LC spots were analyzed using different rastering speeds ranging from 0.25 to 4 mm/sec with the shortest analysis time of 425 ms/spot. Since the LC runs can be multiplexed and do not need a comprehensive investigation, the present platform offers a valuable alternative to LC-ESI-SRM/MS for high throughput proteomic analyses. In addition, the derivatization of the N-terminal α-amino group by sulfonation was found to be key for the fragmentation of singly charged peptides under low collision energy regime. Under such conditions, y-ion series were observed in the MS/MS spectra, and thus the design of SRM experiments was greatly simplified. The quantitative performance of the platform was compared to that of LC-ESI-SRM/MS by spiking yeast tryptic peptides in human plasma digests. Both platforms exhibited similar sensitivities, accuracy (within ±20%) and precision (under 20%) in the relative quantification mode. As a proof of principle, the relative and absolute quantification of proteins associated with glycolysis, glyoxylate and tricarboxylic acid (TCA) cycles over a growth time course of Saccharomyces cerevisiae on glucose media was successfully performed using isotopic dilution.