Objectives: To determine the laboratory based drug abuse prevalence of persons who have illicit and/or addictive substance test requests instead of alcohol in a city in Middle Anatolia in Turkey. Methods: Laboratory information systems data of urine samples, submitted to Biochemistry Laboratory from various clinics, between January 2014 and June 2016 were retrospectively investigated. In comprehension of illicit drug use amphetamin, cannabis, opiate, cocaine and benzodiazepin group tests were screened with Cloned Enzyme Donor Immunoassay method. Positivity-negativity rates according to years, age-gender distribution and number of multiple substance usage were investigated. Results: Totally 79873 illicit drugs were screened in 19763 urine samples. 18080 males (% 91.5) and 1683 females (%8.5), with a mean age of 30.7±10.8 years (range: 4-86) and 27.2±10.5 years (range: 1-86) respectively were included. The average positivity rate according to the sample size in last 2.5 years was 23%; 19% in 2014, 23% in 2015, and 27% in 2016. The positivity rates according to number of analyzed tests were 5.9%, 6.7% and 7.5% respectively. In 2014 the maximum positivity rate was in benzodiazepine (12.5%) and secondly in cannabinoids (11.9%). Whereas amphetamine were the most common drug used (16.3%) with a 4.17 fold increase in 2015 compared to 2014. The increase in amphetamine group substance positivity continued in the first half of 2016 compared to previous year. The most common and the second most common multiple substance usage were amphetamine+ cannabis and amphetamine+opiate respectively. Conclusion: According to laboratory information of illicit drug screening results in urine, drug abuse is increasing through years. Amphetamine group is the most common used substance in the last two years in this region. This is the first study contributing to the prevalence of substance use in a city of Middle Anatolia in Turkey with a large sample volume and patient population.