PyroSmart NextGen^® is a recombinant cascade reagent (rCR) for the detection and quantification of bacterial endotoxins developed using cloned genes derived from the Limulus polyphemus horseshoe crab genome. Requirements for use of this alternative reagent include analysis of analytical performance, method suitability, and test result equivalency to Limulus amoebocyte lysate (LAL) reagents used in the compendial Bacterial Endotoxins Test (BET). The plate reader evaluation has been expanded to address two long-standing user preferences, with the inclusion of the tube reader method increasing the sensitivity of endotoxin detection from 0.005 EU/mL to 0.001 EU/mL. The utilization of PyroSmart NextGen^® with the two different instrument types also allows for a more comprehensive equivalency analysis. Furthermore, the comparison results demonstrate that PyroSmart NextGen^® detects equivalent levels of autochthonous endotoxin in water samples when compared to LAL reagents. Overall, this study provides the first large-scale example of equivalency analysis utilizing a robust rCR and has verified that PyroSmart NextGen^® meets the expectations for alternative reagents.