The uncertainty and sensitivity analysis are evaluated for their usefulness as part of the model-building within Process Analytical Technology applications. A mechanistic model describing a batch cultivation of Streptomyces coelicolor for antibiotic production was used as case study. The input uncertainty resulting from assumptions of the model was propagated using the Monte Carlo procedure to estimate the output uncertainty. The results showed that significant uncertainty exists in the model outputs. Moreover the uncertainty in the biomass, glucose, ammonium and base-consumption were found low compared to the large uncertainty observed in the antibiotic and off-gas CO2 predictions. The output uncertainty was observed to be lower during the exponential growth phase, while higher in the stationary and death phases - meaning the model describes some periods better than others. To understand which input parameters are responsible for the output uncertainty, three sensitivity methods (Standardized Regression Coefficients, Morris and differential analysis) were evaluated and compared. The results from these methods were mostly in agreement with each other and revealed that only few parameters (about 10) out of a total 56 were mainly responsible for the output uncertainty. Among these significant parameters, one finds parameters related to fermentation characteristics such as biomass metabolism, chemical equilibria and mass-transfer. Overall the uncertainty and sensitivity analysis are found promising for helping to build reliable mechanistic models and to interpret the model outputs properly. These tools make part of good modeling practice, which can contribute to successful PAT applications for increased process understanding, operation and control purposes. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1043-1053, 2009