For algae-based biofuel researches, it is usually necessary to extract and quantitatively measure total lipids. The most popular total lipid determination methods for tissues are solvent-based methods with either manual or Soxhlet extraction. Although some investigators have modified these traditional methods, the modifications have rarely been specified in detail, nor has their effectiveness been critically evaluated on microalgae biomass. As a result, there are often significant differences in the analyses of total lipid contents among investigators and laboratories. To assess the effectiveness of different total lipid extraction protocols for microalgae, several conventional lipid extraction methods were systematically compared with an accelerated solvent extraction (ASE) method. Optimal conditions for the ASE method were determined to be three cycles of 3 min at 125 °C using a 5 ml extraction cell containing 20 to 150 mg of dry algal biomass. Critical comparisons and assessments of these total lipid extraction methods from microalgae were performed for both total lipid extraction efficiency and extracted lipid quality (lipid decomposition and oxidation). Results obtained from five algal species indicated that hot Soxhlet extraction and ASE were comparable in total lipid extraction efficiencies, which were higher than the efficiencies obtained from conventional manual extraction methods. However, lipid oxidation resulting from different extraction methods indicated that continuous heating with a hot Soxhlet extraction method could lead to lipid oxidation. Considering the efficiency and quality of the extracted lipids, extraction time, extraction steps and consumed solvent volumes, ASE is a very promising method for total lipid extraction and analysis of lipids from microalgal biomass.