Bioanalytical Pharmacokinetics (PK) methods are designed for robust performance under rigorous regulatory compliance requirements to ensure the generated data is reliable and maintains integrity. In a phase 1 dose-finding clinical study, aberrant PK profiles of two co-administered biologics drugs were observed. Unexpectedly, we discovered high fill levels in collection tubes from the majority of samples. This led to the hypothesis that the highly filled tubes might cause difficulty in achieving complete sample thaw and thorough mixing at the time of sample analysis, potentially contributing to the abnormalities observed in the PK dataset. Evaluation of the impact of sample fill levels and processing procedure can be challenging since PK concentrations of study samples were unknown. Therefore, a systematic approach was employed to conduct a thorough examination using mock samples. The data illustrate a correlation between sample thawing and mixing process and the variability in the PK data. The concentrations from properly filled mock samples that underwent complete thawing and mixing showed 100% data reproducibility. In contrast, the concentrations from fully filled mock samples that did not follow the proper procedure showed sample recovery deviating by ± 30% from nominal value and exhibited considerable lack of precision. This data identified the root cause of aberrant PK, justifying revised sample preparation guidance and sample re-assay. Improved sample handling and subsequent reassay resolved the aberrant PK profile issues. In conclusion, this study reiterated that sample handling plays a crucial role in quality and reproducibility of PK data with immunoassays.