The use of microfluidics has become widespread in recent years because of the use of lesser resources such as small size, low volume of reagents, and physiological representation of mammalian cells. One of the advantages of microfluidic-based cell culture is the ability to perfuse culture media which tends to improve cellular health and function. Although measurement of cellular function conventionally is carried out using well-plates and plate readers, these approaches are insufficient to carry out in-line analysis of perfused cell cultures because of mismatch between volumes and sensitivity. We report the development of a novel microfluidic device and assay that is carried out under perfusion, in-line to measure the cholesterol secreted from a human hepatocyte tissue-chip. The heart of the assay is the unique implementation of enzymatic chemistry that is carried out on a polystyrene bead. Using this approach, we successfully measured cholesterol secreted by the perfused human hepatocytes.