Absorbance measurements via transmitting light spectroscopy in microtiter plates are established for high throughput screening of biological systems. These measurements allow for the determination of important process parameters within a short time. However, absorbance determination via transmitted light measurements is not always feasible. As for carbon monoxide difference absorbance spectroscopy, used for concentration measurements of active P450 monooxygenases (P450s), security standards, and consistent gassing have to be addressed. In this study, a non-invasive online measuring principle for absorbance via scattered light is proposed. Based on optical fiber measurements, a decrease in scattered light signals at 450nm wavelength of reflecting polymer particles is observed, and P450 concentrations are calculated. In this way, high throughput determination of P450 concentrations in a secure, gas-tight environment is realized. The designed method was successfully applied to concentration measurements and carbon monoxide (CO) saturation kinetics ranging from 0.3 to 5.0M P450 BM3 achieving a measurement accuracy of +/- 0.05M P450. Biotechnol. Bioeng. 2017;114: 929-933. (c) 2016 Wiley Periodicals, Inc.