Objective: In this study, it was aimed to determine the anxiety levels of mothers associated with newborn hearing screening test and heel prick blood sampling. Methods: A randomized controlled trial of 112 mothers who applied for newborn screening in a public hospital were conducted. The mothers were divided into two groups depending on their baby's assigned group; hearing screening test group (HST group) or heel prick blood sampling group (HBS group). The study was completed with a total of 101 participants. Parental Information Form, Subjective Units of Distress Scale (SUDS), State-Trait Anxiety Inventory (STAI-S, STAI-T) were used in data collection. Data analysis were performed by using descriptive statistics, Mann-Whitney U test, Kruskal Wallis test, Independent groups t test, Pearson and Spearman correlation. Results: In the HST and HBS groups, the score median of the SUDS was 5,0 (IQR: 4,0-7,0) and 5,0 (IQR: 4,0-7,0), the score average of the STAI-S was 39,8±6,7 and 41,3±7,6, and the score average of the STAI-T was 42,5±7,1 and 41,4±6,5, respectively. There were significant relationships between the mothers' scores of the SUDS, STAI-S and STAI-T. Conclusion: The mothers' anxiety associated with hearing screening test and heel prick blood sampling were higher than Spielberger's female population. There was no difference between the anxiety of the mothers according to whether the screening test is interventional. In order to avoid problems associated with parental anxiety, it may be advisable to investigate appropriate methods to reduce the anxiety of mothers.