Sepsis, a life-threatening condition triggered by infection, has a long history evolving from ancient references to modern understanding. Despite advancements, its symptoms often resemble those of other illnesses until late stages, delaying recognition. The blood culture method, currently used for diagnosing sepsis, is not entirely reliable and often time-consuming, posing challenges for timely intervention in many cases. Recently, miRNAs have emerged as promising biomarkers for a variety of infectious diseases, prompting the suggestion of a new multi-marker panel for sepsis diagnosis. However, the lack of optimized methods for miRNA extraction and quantification necessitates investigation before constructing such a panel. This project aimed to examine and compare manual and robotic methods to determine which yielded superior quality, quantity, time management, and results when employing two-tailed RT-qPCR. Following experimentation, it was observed that both methods provided satisfactory purity and concentration, yet the processing time was notably shorter with the robotic method. The twotailed RT-qPCR method successfully detected DNA with copy numbers as low as 10^5 when spiked-in, although the candidate miRSeps-6 was not detectable in healthy donor plasma. Despite the unsuccessful detection of the candidate miRNA, positive controls demonstrated that processes such as manual and robotic extractions, along with two-tailed RT-qPCR, could be applied to other candidates to identify suitable matches for the kit.