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Introduction The collection of blood samples in different vacutainers can affect the result of serum lithium estimation due to the presence of distinct additives in the blood collection vacutainer for enhancing the clot formation process. Due to the low therapeutic index and threat of toxicity of lithium, it is imperative to correctly report the test result. Thus, it has become a challenge for the laboratory physician to estimate lithium in any clinical laboratory setup. Materials and Methods Sample of 100 patients were collected and paired into clot activator vacutainers and plain glass vials. After centrifugation, samples from the paired collection tubes were processed immediately for serum lithium estimation by VITROS 4600 analyzer working on the principle of reflectance photometry. Both the paired tubes were stored at 2 to 8°C and were further analyzed, at 24 and 48 hours, respectively, from the time of their collection. The statistical analysis was done in IBM SPSS software version 23. Results There was a statistically significant differences between the mean of lithium values when processed within 1st hour of collection, obtained from clot activator vacutainers in comparison to glass vials. However, within tube comparison, there was no statistical difference in the lithium values estimated at 1st hour, 24 hours, and 48 hours of collection. Conclusion In this study, lithium values measured by clot-activated vacutainers are found to be lower as compared with values measured through glass vials. [ABSTRACT FROM AUTHOR]