The use of serum separator tubes has greatly increased efficiency in the laboratory and has reduced in vitro effects on biochemical results. Serum separator tubes have also been associated with interferences regarding endocrine assays and monitoring of therapeutic drug levels. This study measured canine serum cortisol and total thyroxine (TT4) values from both plain serum tubes and serum separator tubes to evaluate for any interference using the Immulite 1000 chemistry analyzer. Linear regression and Bland-Altman analysis demonstrate good to excellent agreement between results from both tube types and no bias for the T4 samples. Cortisol results demonstrate good agreement and minimal negative bias that increased slightly over time. These results indicate that either tube can be used confidently to collect samples for T4 assays using the Immulite 1000. Until canine specific bias levels can be established, SST should be used with caution for baseline cortisol testing in dogs.