AbstractShake flasks are ubiquitous in cell culture and fermentation. However, conventional devices for measuring oxygen concentrations are impractical in these systems. Thus, there is no definitive information on the oxygen supply of growing cells. Here we report the noninvasive, nonintrusive monitoring of dissolved oxygen (DO) in shake flasks using a low‐cost optical sensor. The oxygen‐sensitive element is a thin, luminescent patch affixed to the inside bottom of the flask. The sensitivity and accuracy of this device is maximal up to 60% DO, within the range that is critical to cell culture applications. By measuring actual oxygen levels every 1 or 5 min throughout the course of yeast and E. coli fermentations, we found that a modest increase in shaker speed and a decrease in culture volume slowed the onset of oxygen limitation and reduced its duration. This is the first time that in situ oxygen limitation is reported in shake flasks. The same data is unattainable with a Clark type electrode because the presence of the intrusive probe itself changes the actual conditions. Available fiber optic oxygen sensors require cumbersome external connections and recalibration when autoclaved. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 594–597, 2002.