A novel, simple, rapid, widely applicable, and reliable derivative spectrophotometric technique for the simultaneous determination of enrofloxacin and tylosin has been developed. Enrofloxacin and tylosin belong to the category of fluoroquinolone and macrolide antibiotics, which are widely used in veterinary medicine for poultry. Residues of antibiotics in the edible tissues of food-producing animals arise due to several reasons, such as not adhering to the withdrawal period before consumption, overdosing, and use of long-lasting drugs. First-order derivative spectrophotometry was used for simultaneous determination of enrofloxacin and tylosin in the concentration range of 0.3-31.0 and 0.5-77.5 , respectively. Measurements were performed at 290.69 and 347.02 nm using the zero-crossing technique and at 281.62 nm with the peak-to-baseline technique for enrofloxacin. Zero-crossing wavelengths with amplitude at 272.55, 298.33, and 322.2 nm, and the peak-to-baseline wavelength at 274 nm were used for measurements to determine tylosin. The method was applied for the determination of these antibiotics in different chicken muscle, liver, and kidney samples. The recovery percentages were in the range of 93.01-103.67%.