A classic analytical problem is the simultaneous determination of two or more compounds in the same sample without previous chemical separation. In this way derivative spectrophotometry is an analytical technique of great utility and offers greater selectivity than normal spectrophotometry. The resolution of binary mixtures of compounds with overlapped spectra by derivative spectrophotometry is frequently made on the basis of zero-crossing measurements. A very simple spectrophotometric method using measurements at zero-crossing wavelength is described for resolving binary mixtures of the synthetic colorants sunset yellow (E-110) and erythrosine (E-127). The assay procedure for E-110 and E-127 in Premarin tablets involves extraction of the colorants from tablets with distilled water and measurement of first derivative absorbance values at 482.7 nm for E-127, then filtration from rough paper and measurement of 1 D values at 526.3 run for E-110. Linear correlations over the concentration ranges of 2.0-10.0 mug mL(-1) for E-110 (r = 0.9997) and for E-127 (r = 0.9999) were obtained. Commercially available tablets containing E-110 and E-127 were analysed by the proposed method and the HPLC method. Mean values and standard deviations calculated from seven determinations were 122.5 mug/tablet (RSD = 1.9) of E-110, 119.3 mug/tablet (RSD-2.3) of E-127 for the proposed method and 124.3 mug/tablet (RSD-0.98) of E-110, 120.9 mug/tablet (RSD = 1.2) of E-127 for the HPLC method. The statistical evaluations indicated that there was no significant difference between the mean values and precisions of the two methods at 95% confidence level (t = 1.68 and 0.24 F = 3.76 and 3.67). This method was satisfactorily used for determining synthetic mixtures of these dyes in different ratios. The average percentage recoveries are 105.8% and 102.8% for E-110 and E-127 respectively.