Background: Most of the time pain is difficult to assess and manage because of being inherently a subjective experience influenced by multiple factors. The perception and tolerance of pain may vary because of different psychological and social influences of the patient. Therefore it is important for health care providers to assess the pain so that individualized management interventions can be provided. This study was aimed to assess the nurses’ knowledge and practices related to pain assessment in critically ill patients. Methods: A descriptive cross-sectional study was carried out among 200 registered nurses working at different ICU in Square Hospital. The study was conducted within April to August 2017. A non-probability purposive sampling technique was used. The data was collected using pre-tested self-administered semi-structured questionnaire and it was analysed using SPSS 22.0 version. Results: About 81% of the respondents were in the age group 22-32 years and the mean age of the respondents was 27.74±12.06 years. About 79.0% of the respondents were female and 59.5% of the respondents had diploma in nursing. The mean monthly income of the respondents’ was 19450.5 taka. The majority (59.5%) of the respondents had less than 2 years’ service experience. About 85.5% of the respondents said it is important to assess the pain and need for analgesia before, during, and after wound care. Conclusions: Our findings reported that the nurses were reasonably knowledgeable about the principles of pain assessment. More than four-fifths of the respondents had adequate knowledge about pain assessment.