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Theophylline, salbutamol sulfate and glycerylguaiacolate are active substances for treat asthma (dyspnoea). Combination theophylline, salbutamol sulfate and glycerylguaiacolate in drug preparation have to produce pharmacological effect, so the drug preparation needs the quality assurance of product. This study aims to determine theophylline, salbutamol sulfate and glycerylguaiacolate using ultraviolet spectrophotometry derivative. Research has been conducted in syrup was analyzed using zero crossing wavelength method. NaOH 0,1 N was selected as the solvent. Zero crossing wavelength of theophylline is 275 nm on the first derivative, salbutamol sulfate 262 nm on the second derivative and glycerylguaiacolate 243 nm on the second derivative. Results showed that the recovery of theophylline and glycerylguaiacolate are between 112,913%-118,353% and 116,129%-117,655%, respectively, while the relative standard deviation between 1,090%-1,903% for theophylline and 1,013%-1,922% for glycerylguaiacolate. Limit of detection of theophylline and glycerylguaiacolate are 1,626 ppm and 6,375 ppm, respectively while limit of quantification are 4,927 ppm and 19,319 ppm. So, it concluded that this method could be applied to determine theophylline and glycerylguaiacolate in syrup mixture.