Reliable quantitative description of light-limited growth in microalgae is key to improving the design and operation of industrial production systems. This article shows how the capability to predict photosynthetic processes can benefit from a synergy between mathematical modelling and lab-scale experiments using systematic design of experiment techniques. A model of chlorophyll fluorescence developed by the authors [Nikolaou et al., J Biotechnol 194:91–99, 2015] is used as starting point, whereby the representation of non-photochemical-quenching (NPQ) process is refined for biological consistency. This model spans multiple time scales ranging from milliseconds to hours, thus calling for a combination of various experimental techniques in order to arrive at a sufficiently rich data set and determine statistically meaningful estimates for the model parameters. The methodology is demonstrated for the microalga Nannochloropsis gaditana by combining pulse amplitude modulation (PAM) fluorescence, photosynthesis rate and antenna size measurements. The results show that the calibrated model is capable of accurate quantitative predictions under a wide range of transient light conditions. Moreover, this work provides an experimental validation of the link between fluorescence and photosynthesis-irradiance (PI) curves which had been theoricized.