Phthalates like endocrine disrupting compounds are ubiquitous environmental contaminants. Long term exposure to these chemicals at the work place has adverse effect on human health and usually leads to diseased conditions. The aim of our study was to assess, by biological monitoring, occupational exposure to di-(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DnBP), diethyl phthalate (DEP) and diisononyl phthalate (DiNP) of occupationally exposed group i.e. workers from plastic manufactory with division of films and composites and injection moulding (n=37; average age 45.6±9.4) in comparison with general population i.e. students and workers from Constantine the Philosopher University in Nitra (n=144; average age 28±13.12) in Nitra region of Slovakia. In response to determine human exposure to phthalates, we used high performance liquid chromatography and tandem mass spectrometry analysis (HPLC-MS/MS) to estimate trace levels of 4 phthalate metabolites mono-(2-ethylhexyl) phthalate (MEHP), monoisononyl phthalate (MiNP), mono-n-butyl phthalate (MnBP) and monoethyl phthalate (MEP) in human urine. Median concentrations of the urinary metabolites in the exposed group were 35.48 μg/l for MEHP, 13.65 μg/l for MiNP, 93.79 μg/l for MEP, 108.62 μg/l for MBP and in general population were 21.62 μg/l for MEHP, 13.26 μg/l for MiNP, 84.36 for μg/l MEP and 88.99 μg/l for MBP. We observed, significantly higher urinary concentration (p≤0.001) of MEHP and on the border of the statistical significance urinary concentration of MiNP (p=0.07) in occupationally exposed group. Despite the fact that phthalates are ubiquitous environmental contaminants, there is evidence of occupational exposure of workers from plastic manufactory by DEHP a DiNP. This study was supported by project VEGA (V1/0042/12). © 2014, Mediterranean Center of Social and Educational Research. All rights reserved.