The present study was experimentally conducted to compare the effects of open and closed system of aspiration on pain in newborns given mechanical ventilation. Study population comprised the babies hospitalized in the Newborn Intense Care Unit at Eskisehir Osmangazi University Medicine Faculty Hospital and study sample comprised 42 eligible babies hospitalized in NICU from December 2010 to December 2011. With random sampling, 20 babies were included to the closed-aspiration-system group and 22 to the open-aspiration-system group. Aspiration process was recorded with a camera system. Intervention monitoring form that include oxygen saturation and vital findings, Data collection Form that includes the personal characteristics of the babies, and Newborn Pain/Agitation and Sedation Scale (N-PASS) that evaluate the pain response of the babies were used for data collection. Personal characteristics of the babies were acquired by an investigator from their medical records. Camera records were evaluated by two independent persons, the investigator and a newborn nurse, by using for the NPASS scores. Computerized data were analyzed with using percentile, mean, Standard deviation, chi-square, Student’s-t, matched-t, Wilcoxon-Z, Mann-Whitney-U and Kruskall-Wallis tests. Results of the present study show no statistically difference between the experimental and control groups (p=0.194). N-PASS pain scores were significantly different between pre-intervention period and during the intervention in both groups (p<0.001). In conclusion, we suggest that babies experience pain during the aspiration and although statistically indifferent, an open system of aspiration produces a somewhat higher pain compared the closed system of aspiration.