A dynamic physics-based model developed for the prediction of biohydrogen production in a compact tubular photobioreactor (PBR) was calibrated experimentally. The spatial domain in the model was discretized with lumped control volumes and the principles of classical thermodynamics, mass, species, and heat transfer were combined to derive a system of ordinary differential equations, whose solution was the temperature and mass fraction distributions across the entire system. Two microalgae species, namely, Acutodesmus obliquus and Chlamydomonas reinhardtii strain cc125, were cultured in triplicate with different culture media via indirect biophotolysis. Measured biomass and hydrogen concentrations were then used to adjust the specific microalgae growth and hydrogen production rates in the model based on residual sum of squares (RSS) and the direct search method. Despite its simplicity, the presented volume element model was verified to well predict both hydrogen and biomass concentration over time. The microalgae growth rate for each species was determined as 2.16 μalga,0 s−1 and 0.91 μalga,0 s−1 for A. obliquus and C. reinhardtii strain cc125, respectively, where μalga,0 is the specific growth rate of Scenedesmus almeriensis for given temperature and irradiance. The adjusted maximum hydrogen production rates for the local nonmutant A. obliquus and for C. reinhardtii strain cc125 were 1.3 × 10−7 s−1 and 4.1 × 10−7 s−1. Consequently, these hydrogen production rates were 59 times and 19 times smaller, respectively, than that for the mutant C. reinhardtii strain cc849.