Abstract: The present study was conducted to evaluate the extent of β - lactam antibiotics residues in unprocessed market milk during the year 2006. Milk samples were randomly collected from Hyderabad city, Latifabad and Qasimabad. Microbial screening test (Bacillus subtilus Field Disc Assay) and High Performance Liquid Chromatography (HPLC) methods were used to detect, identify and quantify the β-lactam residues in milk. A total of 137 milk samples were screened. Among these 63.50% were negative and 36.50% positive for β - lactam antibiotics residues. The zones size of positive samples appeared between 5.0 and 15.0mm (mean 8.91 ± 0.36mm). Residues level quantified between 0.4 to 400 µg/L for Penicillin G, between 1.0 to 190µg/L for Amoxicillin, between 0.5 to 141µg/L for Ampicillin and between 2.1 to 122µg/L (40.74 ± 10.59µg/L) for unknown antibiotics. The residues of Penicillin G (mean 59.53µg/L) in unprocessed milk was 14.9 and 11.9 fold, Amoxicillin (mean 36.11µg/L) 9.03 and 3.61 fold, Ampicillin (mean 46.91µg/L) 11.73 and 4.69 fold higher than (MRL`s) standards of EU (4µg/L) and FDA (5 and 10µg/L), respectively.