Background: Family physicians perceive that gut feelings, i.e. a 'sense of reassurance' or a 'sense of alarm', play a substantial role in diagnostic reasoning. A measuring instrument is desirable for further research. Our objective is to validate a questionnaire measuring the presence of gut feelings in diagnostic reasoning. Methods: We constructed 16 case vignettes from real practice situations and used the accompanying 'sense of reassurance' or the 'sense of alarm' as reference labels. Based on the results of an initial study (26 family physicians), we divided the case vignettes into a group involving a clear role for the sense of reassurance or the sense of alarm and a group involving an ambiguous role. 49 experienced family physicians evaluated each 10 vignettes using the questionnaire. Construct validity was assessed by testing hypotheses and an internal consistency procedure was performed. Results: As hypothesized we found that the correlations between the reference labels and corresponding items were high for the clear-case vignettes (0.59 - 0.72) and low for the ambiguous-case vignettes (0.08 - 0.23). The agreement between the classification in clear sense of reassurance, clear sense of alarm and ambiguous case vignettes as derived from the initial study and the study population's judgments was substantial (Kappa = 0.62). Factor analysis showed one factor with opposites for sense of reassurance and sense of alarm items. The questionnaire's internal consistency was high (0.91). We provided a linguistic validated English-language text of the questionnaire. Conclusions: The questionnaire appears to be valid. It enables quantitative research into the role of gut feelings and their diagnostic value in family physicians' diagnostic reasoning.