Microbial cell immobilization has long been considered as a potential bioprocessing strategy to increase both microorganisms' tolerance and fitness in fermentation systems. To date, little emphasis has been put on how the entrapped cells respond to the bioprocessing stresses encountered during the cultivation. The present work presents for the first time a methodology to decipher the real health status of the entrapped microorganisms by combining multiparameter flow cytometry with confocal fluorescence microscopy as monitoring tools. Comparison between resting free and immobilized cell-based systems enabled to characterize the spatial-temporal physiological response of entrapped Pseudomonas taetrolens cells during lactobionic acid production in submerged cultivation. Whereas cellular leakage from beads led to planktonic cells that faced a progressive loss of membrane integrity, immobilized cells underwent a prompt stress-induced physiological response featured by the predominance of cellular damaging. Moreover, visualization without matrix de-entrapment through confocal fluorescence microscopy revealed the overtime formation of cellular micro-colonies inside the beads. These micro-colonies comprised a shell made of dead cells, whereas the inward cells remained metabolically active. The proposed approach herein raises the possibility of using flow cytometry and confocal fluorescence microscopy as indicators of microbial cell immobilization, providing further key information on the health status and robustness of entrapped microorganisms.