Mass spectrometry imaging (MSI) can be used for the µm scale mapping of target molecules on intact sample surfaces. Recently we demonstrated that MSI of non-disturbed sediments can be used for paleoenvironmental studies; using matrix-assisted laser desorption/ionization coupled to Fourier transform-ion cyclotron resonance-mass spectrometry we visualized the spatial distributions of archaeal glycerol dibiphytanyl glycerol tetraether (GDGT) lipids. There is a pressing need for implementing sample preparation procedures that allow exploiting the full potential of sediment MSI. Here we present a suite of sample preparation steps, optimized for the analysis of GDGTs in marine sediments. It considers the crucial requirements for successful MSI and optional combination with elemental imaging via micro X-Ray Fluorescence Spectroscopy (µ-XRF). Preservation of the sediment’s spatial distribution is achieved with freeze-drying and subsequent embedding in a mixture of gelatin and carboxymethyl cellulose. This enables sectioning the sample into sequential slices from 20 to 500 µm in thickness. Thinner sections showed enhanced signal intensity in MSI, but elemental mapping by µ-XRF is more accurate for thicker sections; 100 µm thick slices provide satisfactory results for both analyses and are recommended for congruent elemental and biomarker imaging. When applied to the uppermost ∼5 cm of marine sediment from a Santa Barbara Basin box core, the optimized sample preparation yields reproducible ultra-high-resolution GDGT records from sequential slices, thus demonstrating the robustness of the method. Congruent µ-XRF results aid the establishment of a contextual framework regarding supply of terrigenous and marine detritus as well as the assignment of molecular data to annual layers.