Measuring viability is an important and necessary assessment in studying microorganisms. Several methods can be applied to Leptospira spp, each with advantages and inconveniencies. Here, we describe the traditional colony-forming unit method, together with two other methods based respectively on the reducing capacity of live cells (Alamar Blue Assay) and differential staining of live and dead cells (LIVE/DEAD BacLight). The Alamar Blue Assay uses the blue reagent resazurin, which can be reduced into the pink reagent resorufin by live cell oxidoreductases. Production of resorufin can be quantified by absorbance or fluorescence reading. The LIVE/DEAD BacLight assay uses a mixture of two nucleic acid dyes (Syto9 and propidium iodide) that differentially penetrate and stain nucleic acid of cells with decreased membrane integrity. The colony-forming unit method is labor-intensive but the most sensitive and linear method. The two other methods are not laborious and well-adapted to high throughput studies but the range of detection and linearity is limited.