Number of active smokers in Malaysia is increasing despite availability of stop smoking clinics and smoking cessation medications. Thus, the practice of the healthcare professionals involved in providing smoking cessation intervention using evidence-based guidelines needs to be assessed using validated assessment tool. Newly invented reliable and valid questionnaire locally is needed. This study aimed to develop and validate a newly modified questionnaire to assess the knowledge, attitude and practice of medical doctors based on national smoking cessation guidelines and factors contributing to the score. The 22 items consists of true/false questions and Likert scaling response based on domain of 5A’s (ask, assess, advice, assist, arrange) and 5R’s (relevant, risks, rewards, roadblocks, repetitions) of the national stop-smoking guideline. Sample size is calculated based on five respondents to one item ratio. The questionnaires were distributed to 131 primary care doctors. Reliability was determined using Cronbach’s alpha for internal consistency while construct validity was assessed using factor analysis. A high degree of internal consistency was observed (Cronbach’s alpha = 0.87), with good knowledge subscale (0.72), attitude subscale (0.58) and practice subscale (0.92). Factor analysis showed three meaningful finalized components (knowledge, attitude and practice) which represent the smoking cessation framework. This study indicates that this revised questionnaire is more reliable and valid tool to assess the knowledge, attitude and practice on stop smoking guidelines compared to previous questionnaire.