The Bacterial Endotoxins Test (BET) uses Limulus amoebocyte lysate (LAL) reagents derived from the blood of horseshoe crabs for detection and quantification of endotoxins from Gram-negative bacteria in parenteral drug products and medical devices. Two types of recombinant reagents using genes cloned from the horseshoe crab genome have become available from several suppliers. One is a recombinant Factor C reagent (rFC), containing only recombinant Factor C, and the second is a recombinant Cascade Reagent (rCR) containing recombinant factor C, recombinant factor B and recombinant proclotting enzyme. Implementation of these recombinant reagents for BET requires validation that demonstrates results equal to or better than those determined by LAL reagents. Previous studies have shown that the PyroSmart NextGen^® rCR meets the analytical performance requirements for both the plate and tube reader testing methods and provides equivalent results when testing samples containing autochthonous endotoxin. This study directly compares PyroSmart NextGen^® to LAL reagent performance when testing a parenteral drug, which is a critical step for end-user implementation of alternative methods. It is the first published demonstration of an approach to the validation of alternative reagents that includes testing of a specific parenteral drug sample, and the data indicates that PyroSmart NextGen^® is more precise when compared to LAL reagents. Relative recovery, linear regression, and Bland-Altman plot analyses also illustrate that PyroSmart NextGen^® results are equal to or better than those determined by naturally sourced LAL reagents. This indicates that PyroSmart NextGen^® is a useful alternative method for quantifying bacterial endotoxins in parenteral drugs.