Background Limited resources available to meet unlimited human needs necessitate the prevention of unnecessary use in the field of health. One of the unnecessary expenditures in health is the use of laboratory tests. The aim of this study is to determine the level of unnecessary laboratory test requests and the risk factors affecting unnecessary use in a university hospital. Material and Methods In the study, the 15 most frequently used biochemistry tests in a university hospital were evaluated retrospectively. The Rational Laboratory Test Request Procedure of the Ministry of Health was used to determine the use of unnecessary tests. Multivariate logistic regression analysis was used to identify factors affecting unnecessary test use. Results The analysis results showed that the ratio of unnecessary use of 15 biochemistry tests was 9.1%, and the cost of unnecessary tests was 584.186,3 TL (114.997,3 $). It was found that the probability of requesting unnecessary tests differed significantly according to the branches of the tests requested, the age and gender of the patients and the academic title of the physicians (p<0.001). Conclusions It is thought that the study will provide important clues in the development of managerial interventions to prevent unnecessary test usage and control costs.