Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) imaging is a surface analysis technique that identifies and spatially resolves the chemical composition of a sample with a lateral resolution of less than 1 μm. Depth analyses can also be performed over thicknesses of several microns. In the case of a painting cross-section for example, TOF-SIMS can identify the organic composition, by detecting molecular ions and fragments of binders, as well as the mineral composition of most of the pigments. Importantly, the technique is almost not destructive and is therefore increasingly used in cultural heritage research such as the analysis of painting samples, especially old paintings. In this review, state-of-the-art of TOF-SIMS analysis methods will be described with a particular focus on tuning the instruments for the analysis of painting cross-sections, and with several examples from the literature showing the added value of this technique when studying cultural heritage samples.