Background: In this study, we aim to examine the effects of pre-analytical factors such as specimen type (serum or plasma), collection and storage conditions, and time, on the results of chemiluminescence immunoassay. Methods: Blood samples were collected from 10 individuals and aliquoted into two sets of K3-ethylenediaminetetraacetic acid (EDTA) and serumseparating tubes (SST) each, for plasma and serum collection, respectively.For all the samples, one set of tubes was centrifuged within 1 hour and other set was centrifuged after 4 hours, followed by cell separation.Chemiluminescence assay was performed for adrenocorticotropic hormone (ACTH), parathyroid hormone (PTH), osteocalcin, C-telopeptide, and insulin at 0, 6, 24, and 48 hours after centrifugation; all the samples were assayed in duplicate. The samples were stored at 4℃ before the assay. Results: The results obtained showed that the levels detected in plasmas were more consistent and stable as compared to serum. After a 6-hour storage at 4℃, a significant decrease was observed in the levels of ACTH and osteocalcin in plasma and serum; whereas, PTH and C-telopeptide levels were stable in plasma but decreased significantly in serum. Insulin levels in serum showed a decrease after a 6-hour storage while the levels in plasma were found to be stable until 24-hour storage. Serum samples separated after 4 hours showed a significant decrease in all hormone levels, while C-telopeptide and insulin levels were stable in plasma samples separated after 4 hours. Conclusions The results were found to be more stable in plasma samples from K3-EDTA tubes as compared to serum samples from SST in the measurement of unstable biological analytes. These results suggest that K3-EDTA tubes are preferable in the specimen collection for assaying biological analytes.