This study involves the development and validation of methods for the analysis of antibiotic drug molecules. Techniques for the extraction of residues of these drugs from biological matrices were investigated and subsequently validated. Extraction of sulphonamide residues from porcine muscle was investigated using on-line dialysis, followed by trace enrichment. Direct UV detection and post-column derivatisation was investigated and methods validated. Matrix solid phase dispersion was investigated for the extraction of penicillin residues form porcine muscle. HPLC separation coupled with UV detection of derivatives of the extracted residues was performed. The determination of aminoglycoside residues from milk and porcine liver was investigated. The chromatographic behaviour of these analytes was studied, followed by analysis of aminoglycoside residues using ion-exchange and immunoaffmity column chromatography. Finally, a HPLC method for the separation of aminoglycosides and related compounds was developed, using MS detection.