Autophagic flux, a crucial cellular process that is often overlooked in its significance for human health, has been linked to various chronic diseases, including cancer, heart disease, and dementia. Despite its importance, direct measurement in humans has been lacking, which has hindered translational efforts. Here, we describe a protocol for the measurement of human autophagic flux in blood. By treating whole blood with chloroquine and isolating peripheral blood mononuclear cells, autophagic flux can be measured by quantifying the autophagy protein LC3BII. This approach preserves individual genetic and nutritional parameters. This method can be used to identify factors influencing autophagic flux in humans, facilitating clinical translation and potentially serving as a biomarker for age-related chronic diseases. In this protocol, we describe detailed methodology, along with risks and limitations of this technique.