Designing photobioreactors correctly is a must for the success of microalgal mass production. Optimal photobioreactor design requires a precise knowledge of photosynthesis dynamics in fluctuating light conditions and hence a method for the measurement of photosynthetic rates in specific light regimes. However, it is not uncommon in literature that experimental protocols used to obtain oxygen generation rates are described ambiguously and the reported rates of photosynthesis vary widely depending on the methodology. Additionally, quite a number of methods overlook certain aspects that can affect the estimated rates significantly, and can therefore affect photobioreactor design. We have developed a method based on oxygen evolution measurements that accurately determines photosynthetic rates under well-defined light regimes. Our experimental protocol takes into account most of the issues that can affect the rates of oxygen generation, such as depletion of nutrients during the measurements and precision of the measurements. We have focused on the basic applications in photobioreactor design and used a dynamic model of photosynthesis to analyze our results and compare them with available published data. The results suggest that our oxygen evolution method is consistent. Biotechnol. Bioeng. 2010;106: 228-237. (C) 2010 Wiley Periodicals, Inc.