Background: Sepsis is one of the most serious and life-threatening clinical conditions of childhood. This study has been designed to evaluate how useful multiplex real-time polymerase chain reaction (PCR) is in the early diagnosis of responsible microorganisms of sepsis and to specify how serial procalcitonin level measurement is helpful to support diagnosis of sepsis. Methods: A total of 79 blood samples from 69 consecutive patients were collected for this prospective study between 01 Sept 2009-29 Feb 2012. Results: In the evaluation of patients who are diagnosed with sepsis out of 69 patients with 79 clinical sepsis, 24 (30.37%) had positive blood culture and 19 (24%) samples were positive for PCR. When blood culture and multiplex real-time PCR results were compared, multiplex real-time PCR had a sensitivity of 75% and specificity of 100%. When the 2 microorganisms that multiplex real-time PCR cannot detect are excluded sensitivity increased to 81.8% and specificity did not differ. Procalcitonin levels on the day sepsis is suspected had a mean level of 13.91 ng/mL (± 49.26), on the 1st day (after 24 hours) the mean level of procalcitonin was 15.05 ng/mL (± 43.95), on the 2nd day (after 48 hours) it was 14.89 ng/mL (± 41.57). Mean procalcitonin levels of 50 children admitted with complaints other than infection and systemic inflammation was 0.06 ng/mL (± 0.04). Conclusions: In conclusion, multiplex real-time PCR test would be useful in the early diagnosis of sepsis. Studying procalcitonin levels is helpful in the early diagnosis of sepsis but does not have any correlation with the isolation of microorganisms in blood culture and survival.