Xenografts are a popular model for the study of the action of new antitumor drugs. However, xenografts are highly heterogeneous structures, and therefore it is sometimes difficult to evaluate the effects of the compounds on tumor metabolism. In this context, imaging mass spectrometry (IMS) may yield the required information, due to its inherent characteristics of sensitivity and spatial resolution. To the best of our knowledge, there is still no clear analysis protocol to properly evaluate the changes between samples due to the treatment. Here we present a protocol for the evaluation of the effect of 2-hydroxyoleic acid (2-OHOA), an antitumor compound, on xenografts lipidome based on IMS. Direct treated/control comparison did not show conclusive results. As we will demonstrate, a more sophisticated protocol was required to evaluate these changes including the following: (1) identification of different areas in the xenograft, (2) classification of these areas (necrotic/viable) to compare similar types of tissues, (3) suppression of the effect of the variation of adduct formation between samples, and (4) normalization of the variables using the standard deviation to eliminate the excessive impact of the stronger peaks in the statistical analysis. In this way, the 36 lipid species that experienced the largest changes between treated and control were identified. Furthermore, incorporation of 2-hydroxyoleic acid to a sphinganine base was also confirmed by MS/MS. Comparison of the changes observed here with previous results obtained with different techniques demonstrates the validity of the protocol.