AIM: To analyze the reasons for screening failure and explore the influencing factors in clinical trial in healthy volunteers, guidance was provided to improve the success rate of screening in the future.clarify the reasons for the failure in healthy subjects (HS) screening, and to provide guidance for screening in phase Ⅰ clinical trials. METHODS: We performed a retrospective study that described the process of HS screening in phase I clinical trials carried out in department of clinical pharmacology lab, Nanjing First Hospital between 2019 and 2022. We analyzed the reasons for screening failure and their impact on the failure rate. A retrospective analysis was conducted on the data of subjects who participated in drug clinical trial screening 2019 to 2022. The reasons for screening failure were analyzed, and statistical methods were used to explore the independent factors that led to screening failure. RESULTS: A total of 11 clinical trials were included in this study, and 502 out of 1 582 participants (31.7%) passed the screening. The analysis of the remaining 1 080 subjects showed that the items that did not pass the screening were laboratory examinations (631 cases, 58.4%), abnormal vital signs results (228 cases, 21.1%), intolerance to blood drawn (86 cases, 8.0%), sufficient subjects (62 cases, 5.7%), withdrawal at the screening (54 cases, 5.0%), demography (54 cases, 5.0%), urinary cotinine examination (42 cases, 3.9%), imaging examination (31 cases, 2.9%), electrocardiogram (24 cases, 2.2%), inquiry (medical inquiry 19 cases, 1.8%，smoking inquiry 2 cases, 0.2%, alcohol inquiry 2 cases, 0.2%) and identity verification (17 cases, 1.8%). In the population with a body mass index (BMI) of 19.0 to 26.0, an increase in BMI is an independent factor significantly associated with screening failure (P<0.000 1, OR=0.890 4, 95%CI 0.841 9-0.941 3). The impact of different examination items on the screening failure rate varies. CONCLUSION: In clinical trials of healthy subjects, laboratory tests, vital signs and intolerance to blood drawn are the main reasons for screening failure. Lowering the upper limit of BMI when recruiting subjects may increase the success rate of screening. Laboratory examinations, vital signs, intolerance to blood drawn are the most important three reasons for screening failure, and improvements can be made to reduce the screening failure rate of phase Ⅰ clinical trials in response to the main screening failure reasons.