A new derivative spectrophotometric (DS) method was proposed and validated for quantification of total flavonoids from in O/W emulsion with polyacrylamide (and) C13-14 isoparaffin (and) laureth-7 containing Catuaba (Trichilia catigua Adr. Juss) (and) Marapuama (Ptychopetalum olacoides Bentham) extract. DS method was optimized to perform the assay in most favorable conditions. Linearity, specificity and selectivity, recovery (Rc, %), precision (R.S.D., %), accuracy (E, %), detection (LOD, microg ml(-1)) and quantification limits (LOQ, microg ml(-1)) were established for method validation. First-derivative at 388.0 nm (zero-to-peak; amplitude= +/- 0.12; wavelength range= 300.0-450.0 nm and Deltalambda = 4 nm) offered linearity for rutin concentrations ranging from 10.0 to 60.0 microg ml(-1) in ethanol 99.5%. Second-derivative provided to be unsuitable for interval evaluation obtaining unacceptable accuracy. Analytical method was validated for first-derivative, according to the experimental results: correlation coefficient (r = 0.9999); specificity to total flavonoids quantification, expressed in rutin, at wavelength 388.0 nm and selectivity with elimination of interference from matrix; Rc = 108.78%; intra- and inter-run precision (1.30-3.65% and 3.48-4.68%), and intra- and inter-run accuracy (100.00-112.19% and 101.25-118.44%); LOD = 0.62 microg ml(-1) and LOQ = 1.86 microg ml(-1).