Polycyclic aromatic hydrocarbons (PAHs) are produced as primary environmental pollutants as a result of incomplete combustion of fuels. They harbor mutagenic and carcinogenic properties. Their continuous monitoring is considered a requisite for keeping them into admissible limits. The current study was designed to quantify the levels of PAHs in soil, water and blood samples. One hundred and fifty samples each of soil, water, human and animal blood were collected from highly exposed areas including industrial, highways and incinerators linked areas around Lahore city, Punjab, Pakistan. Amounts of six selected PAHs (Phenanthrene, Biphenyl, Biphenthrene, Naphthalene, Anthracene and P-Ansidine) were quantified by reverse phase-high performance liquid chromatography (RP-HPLC) equipped with UV-VIS photodiodes array detector (PDA) at 247 nm. An isocratic method was optimized and validated for use with soil, water and plasma samples. To obtain the reliable results the HPLC method was validated following the ICH/FDA guidelines. It was found that PAHs exist in large quantity in highly exposed areas particularly the soil samples were extremely contaminated with PAHs. The highest average concentration of Naphthalene (260.85±165.64 μg/kg) was detected in samples from industrial areas. Conversely, the lowest average amount of Biphenanthrene (10.31±3.46 μg/kg) was found in samples from highways. Human and animal plasma samples were also found to carry PAHs in comparatively lower levels than soil and water.