Entrapped bacteria are used in several applications including food and beverage production, antibiotic production, and wastewater treatment. To date in order to determine the viability of entrapped bacteria, they have to be de-entrapped from the matrix first. However, cell de-entrapment procedures, such as matrix dissolution by acid or heating at high temperatures, may affect the viability of the cells. In this study, three viability assays were assessed for in situ estimation of the number of entrapped cells. Also, a new method was developed to determine the effect of entrapment procedures on bacterial cell viability using one of the three assays, LIVE/DEAD® BacLight™ Bacterial Viability Kit. The other two quantitative assays used in this study were the bioreducible tetrazolium salt (XTT) assay and the adenosine triphosphate (ATP) based assay. The applications of the assays and the new method were performed on two cell entrapment techniques widely used in environmental applications, phosphorylated-polyvinyl alcohol (PPV A) and calcium alginate (CA). The data from the XTT and ATP assays showed linearity and strong correlations between the viability signals and number of beads in which each bead contained a similar number of live cells. An application of the XTT assay on the PPV A entrapped bacterial beads was an exception to these results. Effects of the acid and heat dissolution deentrapment procedures on cell viability were also evaluated by using both assays and a traditional plate count method. The heating process showed the greatest reduction in bacterial viability when compared to the other de-entrapment procedures. The ATP assay is a more sensitive and less time consuming approach for viability estimation when compared to the XTT assay and traditional plate count method. Both XTT and ATP assays have potential for use in quantifying the viability of entrapped bacteria. The new method developed for determining the effect of entrapment procedures on bacterial cell viability involved entrapping bacteria directly onto glass slides. This new method was compared with traditional approaches which require dissolution of the entrapment matrix using chelating agents and heat. Both the developed and traditional methods require labeling with fluorescent dyes from the LIVE/DEAD® assay and observing and quantifying live and dead cells under fluorescence illumination. The viability of entrapped cells was compared to the viability of free cells prior to the entrapment. The developed method was applicable to both PPV A and CA entrapped cells. Both methods indicated that the entrapment procedures resulted in reductions in cell viability, but the new method showed less viability reduction than the previously used method. This suggests that the matrix dissolution prescribed in the traditional method negatively affected cell viability and the new method is therefore more reliable. The percent of live bacterial cells before the entrapment ranged from 54 to 74%, while the percent of live cells following the entrapment based on the new method was 39 to 62%. The approach used in the method could potentially be adopted for other cell entrapment techniques.