BACKGROUND: Behavioural theories are crucial to the development and success of effective hand hygiene interventions. Research demonstrates that hand hygiene behaviours can also be influenced by religious and cultural beliefs. In this paper we detail the processes undertaken to develop a valid and reliable questionnaire to examine healthcare workers' beliefs and behaviours related to the use of alcoholbased hand rub (ABHR), with particular emphasis on associated religious and cultural factors. METHODS: Using the Theory of Planned Behaviour, an existing questionnaire was modified and pilot-tested on 35 staff in the Quality Department. Cronbach's alpha values and mean inter-item correlations of the scale items were the main outcome measures. A Pearson correlation was conducted to assess for social desirability response bias. RESULTS: The questionnaire demonstrated high content and face validity and satisfactory internal consistency in most scales, except for the religious beliefs, cultural beliefs and perceived behavioural control scales, where the inter-item correlations were 0.10, 0.13, and 0.14, respectively. There was a significant correlation between scores on the social desirability scale and the attitude scale. Partial correlation should be used to control the effect of social desirability responding when analysing data from the attitudes scale. CONCLUSIONS: A validation process allowed the introduction and subsequent modification of religious and cultural belief scales to an existing questionnaire examining behavioural and attitudinal influences on hand hygiene using ABHR. Such processes should be considered for questionnaire development and modification when considering religious and cultural factors as drivers of behaviour.