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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, μg ml−1) and quantification limits (LOQ, μg 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 Δλ = 4 nm) offered linearity for rutin concentrations ranging from 10.0 to 60.0 μg 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 μg ml−1 and LOQ = 1.86 μg ml−1.