The objective of this study was to develop a reduced-order model of biodegradation in unsaturated soils that allows the estimation of contaminant depletion, using available on-line measurements. Hexadecane was chosen as a model compound for petroleum hydrocarbons. A two-compartment model was developed, decoupling the intrinsic biodegradation kinetics from limiting factors imposed by field conditions, such as oxygen transfer and contaminant bioavailability. Two new experimental protocols (one for the liquid phase and the other for the solid phase) were developed to monitor hexadecane depletion, hexadecane mineralization, total mineralization, and evolution of the degraders. Using the liquid-phase experiment, parameters of a Haldane kinetic model and yield coefficients were identified and used in the complete model of biodegradation in soil. Using the carbon dioxide production curve, a biocontact kinetic model was identified so that, despite the high sensitivity of the model outputs to variations in the parameters, hexadecane depletion could be correctly predicted with an average error on the entire time trajectory of about 8%. Moreover, the ratio between hexadecane mineralization and total mineralization remained constant after a brief transient period, indicating that hexadecane mineralization could be deduced from the total carbon dioxide measurement. Finally, the new model developed in this study allows real-time monitoring of contaminant biodegradation, using on-line carbon dioxide measurement.