SummaryHere, we present a protocol for assessing metabolic activity of bacterial populations by measuring heat flow using isothermal calorimetry. We outline the steps for preparing the different growth models ofPseudomonas aeruginosaand performing continuous metabolic activity measurements in the calScreener. We detail simple principal component analysis to differentiate between metabolic states of different populations and probabilistic logistic classification to assess resemblance to wild-type bacteria. This protocol for fine-scale metabolic measurement can aid in understanding microbial physiology.For complete details on the use and execution of this protocol, please refer to Lichtenberg et al. (2022).1