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A new procedure for the determination of biogenic amines (BAs), putrescine, cadaverine, spermidine, spermine and tyramine, based on the purification through precipitation and centrifugation of peptides in an acid extract from the sample is proposed. The novelty of the procedure in relation to the liquid-liquid purification reported in the literature is based on shortening the time of analysis and reducing BAs losses during sample preparation. The procedure permitted better precision, recovery, and sensibility, particularly in lower concentration levels, when compared to extraction without purification before derivatization. The method was applied to the simultaneous determination of the five aforementioned BAs in commercial yogurt samples by reversed-phase high-performance liquid chromatography (RP-HPLC). The procedure presented adjusted correlation coefficients between 0.980 and 0.997, and linear range in samples of 1–50 mg L−1 (cadaverine), 3–50 mg L−1 (putrescine, spermidine and spermine) and 5–50 mg L−1 (tyramine), with accuracy ranging from 67.81% to 142.78% in the limit of quantification (LOQ) and from 88.99% to 118.26% in other concentration levels. The limit of detection of BAs in yogurt samples was 0.18–4.01 mg L−1, while LOQ ranged between 1 and 5 mg L−1. The intraday relative standard deviation was ≤15.74% in LOQ and ≤10.05% in other concentration levels. The purification step by precipitation was fundamental to eliminate the interferences present in the yogurt samples, and it was relevant to increase the sensitivity of BAs detection when compared to liquid-liquid purification. Keywords: Extract purification, Sample preparation, In-house validation method, Tyramine, Cadaverine, Putrescine, Fermented milk, FDA Guidelines, Decision limits, Detection capabilities