Theophylline is a drug widely known for treating asthma and sorne other chronic respiratory diseases. At present time, the blood levels of drugs can be related with eficacy and side effects. There are around 10 drug products ofprogrammed release theophyIline forms, commercially available in Colombia. Most of the problems found in the utilization are caused by the non advisable interchange of theophyIline drug products, without any bioequivalency data for them. This study proposes a specific and validated analytical methodology for theophylline in biological fluid s including blood, which is useful for bioavailability and bioequivalency studies, rutinary monitoring of theophylline blood levels and in forensic chemistry. This metodology has the advantage. to use a mobile phase les s contaminant and cheaper than others previously used. Besides, it makes easier the cleaning of the equipment and extend the useful life of the column. The chromatographic separation system by HPLC-RP, with spectrophotometric detection at 266 nm, ineludes an octadecylsilane C-18 column, and methanol/water as mobile phase.