The efficient quantification of bioprocess parameters is of high importance for strain characterization and process development. Here, we present a method, which allows the quantification of the specific growth rate, the specific substrate uptake rate and the growth stoichiometry, in a real-time context. The method is solely based on the combination of first principle relationships, using unstructured kinetic modeling, elemental- and mass balancing. Hence, very little prior knowledge is required. Off gas measurements of CO2 and O-2 as well as base consumption were used as the only input variables to the method, which was executed in real-time along with the bioprocess. We demonstrate the capacity of the method by fed-batch cultivations of the methylotrophic yeast Pichia pastoris as well as by batch cultivations of P. pastoris and Saccharomyces cerevisiae on various carbon substrates. In fed-batch, with an accelerated and decelerated exponential feed profile, the method was able to efficiently quantify the maximum specific growth rate, as well as to investigate growth stoichiometry at low growth rates for maintenance effects. Furthermore, the method allows to correctly identify the timely adaptation of the specific substrate uptake rate and the specific growth rate as well as the oxidative capacity in batch cultures and to distinguish substrate specific kinetics. Further real-time outputs are concentrations of components such as biomass and substrate. The validity of the derived parameters is checked for consistency using redundancy and a statistical test. This method contributes to process understanding, because the method consistently quantifies bioprocess parameters, which can be used to analyze their correlation to critical quality attributes. Furthermore, the real-time availability of the parameters allows the development of process control strategies. This matches Quality by Design (QbD) and Process Analytical Technology (PAT) in its main objectives. (C) 2010 Elsevier Ltd. All rights reserved.