In the last few years, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has been successfully used to study the distribution of lipids within tissue sections. However, few efforts have been made to acquire reliable quantitative data regarding the localized concentrations of these molecules. Here we propose an approach based on brain homogenates for the quantification of phosphatidylcholines (PCs) in brain section by MALDI MSI. Homogenates were spiked with a range of PC(16:0 d31/18:1) concentrations. Sections from homogenates and intact brain were simultaneously prepared before being analyzed by MALDI MSI using a Fourier transform ion cyclotron resonance (FT-ICR) analyzer. Standard curves were generated from the signal intensity of the different PC(16:0 d31/18:1) ionic species ([M+H](+), [M+Na](+) and [M+K](+)) detected from the homogenate sections. Localized quantitative data were finally extracted by correlating the standard curves with the signal intensities of endogenous PC (especially PC(16:0/18:1)) ionic species detected on different areas of the brain section. They were consistent with quantitative values found in the literature. This work introduces a new method to take directly into account biological matrix effects for the quantification of lipids as well as other endogenous compounds, in tissue sections by MALDI MSI.