There is a need to assess the knowledge of healthcare providers on the use of maternal analgesics during lactation, while a valid instrument is not yet available. This study aimed to develop a valid and reliable questionnaire on the knowledge of analgesics (acetaminophen, ibuprofen, aspirin, tramadol, codeine, oxycodone) during lactation, using a prospective, stepwise approach. To generate a pool of item subgroups, literature was assessed as first step. This preliminary version was subsequently reviewed in two focus groups [midwives (n=4), pediatric nurses (n=6)], followed by an expert panel (n=7, 2 rounds) to confirm content validity [item-level and scale content validity]. This resulted in a instrument consisting of 33 questions, and 5 clincial case descriptions specific for both disciplines. Based on known-groups validity between midwives and pediatric nurses (assuming an a priori difference related to their curricula), high construct validity was subsequently demonstrated in a pilot e-survey (86 midwives, 73 pediatric nurses). We therefore conclude that an instrument to assess knowledge on lactation-related exposure to analgesics was generated, that can be further developed and validated. Furthermore, pilot findings suggest suboptimal knowledge for both professions, so that adaptations in their curricula and postgraduate training are warranted.