Non-cholesterol sterols (NCS) are used as surrogate markers of cholesterol metabolism which can be measured from a single blood sample. Cholesterol precursors are used as markers of endogenous cholesterol synthesis and plant sterols are used as markers of cholesterol absorption. However, most aspects of NCS analysis show wide variability among researchers within the area of biomedical research. This variability in methodology is a significant contributor to variation between reported NCS values and hampers the confidence in comparing NCS values across different research groups, as well as the ability to conduct meta-analyses. This paper summarizes the considerations and conclusions of a workshop where academic and industrial experts met to discuss NCS measurement. Highlighted is why each step in the analysis of NCS merits critical consideration, with the hopes of moving towards more standardized and comparable NCS analysis methodologies. Alkaline hydrolysis and liquid-liquid extraction of NCS followed by parallel detection on GC-FID and GC-MS is proposed as an ideal methodology for the bio-analysis of NCS. Furthermore the importance of cross-comparison or round robin testing between various groups who measure NCS is critical to the standardization of NCS measurement.