Lipidomics aim to quantify lipid species in all kinds of samples, including tissues. To subject a fixed amount of sample to various workflows, tissue homogenates were frequently prepared at defined concentrations in water or by addition of organic solvents. Here, we investigated this first step of tissue lipidomics by quantitative flow injection analysis coupled to Fourier-Transform mass spectrometry (FTMS). The influence of sample concentration, solvent composition, and homogenization procedure on the recovery of lipids was studied in murine liver. Liver homogenates were prepared either by grinding tissue in liquid nitrogen or by bead-based homogenization. Ground samples were dissolved at different concentrations in water, methanol, and water/methanol = 1/1 (v/v). Here, lipid recovery depends on solvent composition and sample concentration. The recovery of nonpolar lipid classes, including triglycerides and cholesteryl ester, was decreased in methanolic homogenates. In contrast, due to superior dispersion of precipitates, bead-based homogenization resulted in efficient lipid recovery independent of the solvent composition. However, lipid distribution within samples, i.e., lipid content of supernatant and pellet following centrifugation, was altered substantially by solvent composition. In conclusion, accurate lipid quantification of tissue homogenates requires evaluation of solvent composition, sample concentration, as well as the homogenization method to guarantee efficient lipid recovery. Due to a potential loss of lipids, removal of precipitates by centrifugation prior to lipid extraction should be avoided.