Clinical practice is highly stressful for nursing students. There are various stressors in clinical practice. The purpose of this study was to develop an instrument with acceptable validity and reliability to measure the perceived stress of nursing students in clinical practice. Thirty-three items were initially developed by interviewing 100 nursing students. The subjects consisted of 580 nursing students with clinical practice experience. The instruments include physical-psychological response scale and G-Z temperament schedule. Based on the results of item analysis and factor analysis, 4 of these items were deleted. The remaining 29 items are grouped into 6 factors, including stress from taking care of patients, stress from the faculty of the school and the staff of hospitals, stress from their homework and workload, stress from their peers and in their personal lives, stress from the clinical environment, and stress related to professional knowledge and skills. The results of this study showed that this instrument possesses acceptable content validity, construct validity, criterion validity and internal consistency reliability. In conclusion, this instrument with 29 items and 6 factors is useful, valid and reliable.