Phytosterol measurement has gained a lot of interest during the last two decades after foods and supplements with added 4-desmethyl phytosterols were recognized and used as effective and safe non-pharmacologic hypocholesterolemic agents, and also after the mechanisms of intestinal absorption and hepatic excretion of sterols were unraveled. In addition, the wide use of serum phytosterols as biomarkers of cholesterol absorption has increased the interest in their measurement. In this review, the basic methods are discussed without going into details of the practical operations. The analysis includes first lipid extraction and saponification from various biologic matrices such as serum/plasma, feces, or tissues, after which the individual sterols are separated by adsorption chromatography (gas-liquid or liquid or high performance liquid chromatography) based on the polarity of the various sterols. We also deal with some specific aspects of phytosterol measurements in biological samples such as the need of harmonization of their analysis in biological samples, the discrepancies in the results of sitosterol and campesterol concentrations between different studies, and what is known about their biological day-to-day fluctuation. Phytosterols have a remarkable role in human health, so that their complicated and time consuming measurements call attention to routine ways of standardization between the sterol research laboratories.