Non-invasive, simultaneous optical monitoring of oxygen and pH during bacterial cultivation in 24-well microplates is presented using an integrated dual sensor for dissolved oxygen and pH values. The dual sensor is based on oxygen-sensitive organosilica microparticles and pH-sensitive microbeads from a polymethacrylate derivative embedded into a polyurethane hydrogel. The readout is based on a phase-domain fluorescence lifetime-based method referred to as modified frequency domain dual lifetime referencing using a commercially available detector system for 24-well microplates. The sensor was used for monitoring the growth of Pseudomonas putida bacterial cultures. The method is suitable for parallelized, miniaturized bioprocessing, and cell-based high-throughput screening applications. Biotechnol. Bioeng. 2008;100: 430–438. © 2008 Wiley Periodicals, Inc.