<p>Needle-related procedures are painful for infants, highlighting the need to enhance the use and understanding of non-pharmacological strategies to alleviate pain and anxiety during such procedures. This study aims to explore how needle-related routine care procedures, such as vaccinations and blood samples, are designed and described by parents and healthcare professionals to minimize pain and anxiety in newborns and infants aged 3 and 5 months, in the framework of Kolcaba’s Comfort Theory. Using a convergent mixed-method approach, which combines qualitative and quantitative data collection and analysis, the results are presented according to the three pillars of Comfort Theory: relief, ease, and transcendence. In total, 16 healthcare professionals, 40 parents, and 27 infants aged 0–5 months were included in the study. The findings describe how needle-related procedures were carried out and perceived by healthcare professionals and parents. Several key aspects were identified as important for alleviating pain, including support for parents, pain relief, preparation for parents, support and comfort for the infant, ensuring safety for the infant and experiences of well-being. The study suggests several practices to decrease pain and anxiety for infants undergoing needle-related procedures in routine care. These include supporting and preparing parents with information about the procedure and possible pain relief methods for the infant, fostering a calm environment, and ensuring the safety of the infant throughout the process.</p><p><b>Trial registration</b> ISRCTN 12280133. Registration date: 24/04/2023.</p>