Sedimentary lipid biomarker proxies are extensively used for paleoclimatic and paleoenvironmental research. Among all the lipid compounds, the non-polar n-alkanes (chain lengths n-C14 to n-C37) are one of the most widely used lipid fractions for such studies. However, the lack of optimized automated extraction and post-extraction sample workup procedure, using conventional polar solvent, from environmental samples, has been challenging. Conversely, extraction of the n-alkanes using non-polar solvents restricts the co-extraction of other polar lipid compounds like ketones, alcohols, acids, etc. which may be of interest to many researchers. Here we report an optimized total lipid extraction method using the conventional polar dichloromethane/methanol solvents with standardized Automated Solvent Extractor (ASE) parameters and post-extraction sample work-up protocols. The robustness of the method has been tested by extracting eight samples containing algae, leaf, and sediment/rock of varying age (spanning from recent to ∼252 Ma). The results of the extraction efficiency (yield) of the method were compared with commonly used methods, those using either non-polar n-hexane or polar dichloromethane/methanol solvents. For n-alkane extraction from algal biomass, the performance of the proposed method and the conventional polar solvent method are statistically comparable, but the new method performs better than the non-polar solvent method. Likewise, for the leaf and sediment samples, the performance of the proposed method is equivalent to the non-polar solvent method but outperforms the commonly used polar solvent method. The Terrestrial Aquatic Ratio (TAR) and long-chain hydrocarbon/short-chain hydrocarbon (LHC/SHC) ratios calculated from the algae and sediment samples also show that our recommended extraction approach can be confidently used in calculating n-alkane ratio proxies, which critically depend on the extraction efficiency of the short-chain n-alkanes. Taken together, the new method will be highly useful for studying the n-alkanes from a wide spectrum of natural sample matrices and provide reliable data for paleoclimatic inference.