Glycerol dialkyl glycerol tetraethers (GDGTs), specific membrane lipids synthesized mainly by bacteria and archaea, can be divided into isoprenoids and methyl branched alkyl GDGTs (iGDGTs and brGDGTs, respectively). Three GDGTs groups (iGDGTs, brGDGTs, and other membrane lipids) in a peat sample were separated and collected in this study using semi-preparative high-performance liquid chromatography (HPLC) and silica gel column chromatography. The obtained recoveries for the whole analytical procedure were 85% – 55% using semi-preparative HPLC and 70% – 20% using gel column chromatography. In addition, in each method, the recoveries of brGDGTs and iGDGTs were similar, regardless of the huge difference in their contents. High purity was found in the fractionated groups, determined based on ether cleavage and reduction. Moreover, the semi-preparative HPLC method could realize a better separation efficiency than the silica gel method, but it was time-consuming and required expensive equipment, while the silica gel chromatography method featured merits of time saving and convenient operation at the cost of a slight reduction in separation efficiency. The advantages of the silica gel method make it an operational laboratory method for batch experiments and isotopic studies.