Currently, no fast and accurate methods exist for measuring extant biokinetic parameters for biofilm systems. This article presents a new approach to measure extant biokinetic parameters of biofilms and examines the numerical feasibility of such a method. A completely mixed attached growth bioreactor is subjected to a pulse of substrate, and oxygen consumption is monitored by on-line measurement of dissolved oxygen concentration in the bulk liquid. The oxygen concentration profile is then fit with a mechanistic mathematical model for the biofilm to estimate biokinetic parameters. In this study a transient biofilm model is developed and solved to generate dissolved oxygen profiles in the bulk liquid. Sensitivity analysis of the model reveals that the dissolved oxygen profiles are sufficiently sensitive to the biokinetic parameters - the maximum specific growth rate coefficient (μ) and the half-saturation coefficient (K(s)) - to support parameter estimation if accurate estimates of other model parameters can be obtained. Monte Carlo simulations are conducted with the model to add typical measurement error to the generated dissolved oxygen profiles. Even with measurement error in the dissolved oxygen profile, a pair of biokinetic parameters is always retrievable. The geometric mean of the parameter estimates from the Monte Carlo simulations prove to be an accurate estimator for the true biokinetic values. Higher precision is obtained for estimates than for K(s) estimates. In summary, this theoretical analysis reveals that an on-line respirometric assay holds promise for measuring extant biofilm kinetic parameters.