Background Using standard pain assessment techniques is a cornerstone for effective pain management. Pain is not assessed in a standardized manner in numerous practice settings. The problem of applying pain assessment principles was found to be higher in low-income countries. Very limited evidence indicates the standard of pain assessment practice among nurses in Ethiopia. Therefore, the purpose of this study was to identify pain assessment practice and associated factors among nurses working at adult care units in public hospitals in the Wolaita Zone, Southern Ethiopia. Methods A cross-sectional study was conducted among 290 nurses working at adult care units in public hospitals in Wolaita zone. Data were collected from February to March 2021. A structured self-administered questionnaire was used for data collection. Epi Data 4.6 was used to enter the data, and SPSS version 26 was used to analyze the data. A logistic regression model was used, and statistical significance was declared at P ≤ 0.05. An adjusted odds ratio with 95%CI was used to measure the degree of association. Results A total of 267 nurses participated in the study, giving a response rate of 97.8%. Almost three-fourths (73.8%) of the study nurses reported that they assessed pain for their patients. Only 23.6% of the study nurses discussed pain assessment scores during a nurse-to-nurse report. Below, half (47.2%) of the study participants documented pain assessment scores. The proportion of nurses with good pain assessment practice was found to be 38.2%. The odds of having good pain assessment practice among nurses who received training on pain management was two times higher than its counterpart. The nurses who perceived organizational support were twice more likely to have good pain assessment practice than their counterparts. Conclusion Nurses’ pain assessment practice was found to be low. Moreover, a substantial proportion of the study nurses did not discuss pain assessment scores during a nurse-to-nurse report with low documentation practice. Continuous professional development through in-service training and education is crucial to the improvement of nurses’ pain assessment practice. Furthermore, ameliorating organizational support by means of a supportive working environment is suggested to the betterment of nurses’ assessment practice.