Biofuels produced from lignocellulosic biomass via the fermentation platform are sustainable energy alternatives to fossil fuels. Process Systems Engineering (PSE) uses computer-based tools and methods to design, simulate and optimize processes. Application of PSE tools to the design of economic biorefinery processes requires the development of simulation approaches that can be integrated with existing, mature PSE tools used to optimize traditional refineries, such as Aspen Plus. Current unit operation models lack the ability to describe unsteady state fermentation processes, link unsteady state fermentation with in situ separations, and optimize these processes for competing factors (e.g., yield and productivity). This work applies a novel architecture of commercial PSE tools, Aspen Plus and MATLAB, to develop techniques to simulate time-dependent fermentation without and with in situ separations for process design, analyses and optimization of the operating conditions. Traditional batch fermentation simulations with in situ separations decouple these interdependent steps in a separate “steady state” reactor followed by an equilibrium separation of the final fermentation broth. A typical mechanistic system of ordinary differential equations (ODEs) describing a batch fermentation does not fit the standard built-in power law reaction kinetics model in Aspen Plus. To circumvent this challenge, a novel platform that links the batch reactor to a FORTRAN user kinetics subroutine (incorporates the ODEs) combined with component substitution (to simulate non-databank components) is utilized to simulate an unsteady state batch and in situ gas stripping process. The resulting model system predicts the product profile to be sensitive to the gas flow rate unlike previous “steady state” simulations. This demonstrates the importance of linking a time-dependent fermentation model to the fermentation environment for the design and analyses of fermentation processes. A novel platform linking the genetic algorithm multi-objective and single-objective optimizations in MATLAB to the unsteady state batch fermentation simulation in Aspen Plus through a component object module communication platform is utilized to optimize the operating conditions of a typical batch fermentation process. Two major contributions are: prior concentration of sugars from a typical lignocellulosic hydrolysate may be needed and with a higher initial sugar concentration, the fermentation process must be integrated with an in situ separation process to optimize the performance of fermentation processes. With this framework, fermentation experimentalists can use the full suite of PSE tools and methods to integrate biorefineries and refineries and as a decision-support tool to guide the design, analyses and optimization of fermentation-based biorefineries.