Objectives: Ultrasound (US) has been reported to be a reliable imaging modality for locating the tip of peripherally inserted central venous catheters (PICC) in neonates. However, its use requires a certain level of expertise, which may limit its application. Standardization of US examinations using designed protocols may improve their efficiency and ease of use. The objective of our study is to evaluate the effectiveness of introducing a standardized US protocol for PICC guidance. Methods: An expert panel was formed in order to develop a standardized US protocol for PICC assessment. Institutional review board agreement was obtained. This protocol was then used by participating clinicians to assess PICC position. Every assessment was followed by a radiographic control. The study group consisted of 262 US examinations of PICC lines in a level III neonatal intensive care unit (NICU) performed according to the designed protocol. Subsequent statistical analysis was made with the RStudio software, R version 4.3.1 (2023-06-16 ucrt). Results: Compared to the X-ray control, standardized US examinations showed 100% sensitivity, 81% specificity, an accuracy of 98% and a 98% precision for assessing catheter placement. The Cohen's Kappa value for the comparison of X-ray and US studies was 0.88, indicating good agreement between the two methods. Conclusions: Standardized US examinations have shown similar potential for evaluating the location of PICC lines when compared with radiographic studies. Implementing a standardized protocol in the NICU may facilitate the ultrasound assessment of PICC lines and reduce exposure to ionizing radiation.