Honey production has been compromised due to contamination and the disappearance of bees. Contaminations are usually caused by the use of pesticides in places where pollination occurs and, consequently, affect bees and honey produced. Exposure to the herbicide Glyphosate at sub-lethal doses promoted a lower sensitivity and decreased associative memory in bees. Glyphosate is categorized as non-selective systemic herbicide for post-emergence use. AMPA is its most common metabolite. Glyphosate and AMPA are difficult herbicides in the trace analysis, because they have low molecular weight, low volatility and good solubility in water. They are generally determined by the method established by Cowell et al. (1986) using liquid chromatography with fluorescence detector and post-column derivatization reaction. The post-column reaction step involves oxidation of glyphosate with hypochlorite into glycine and the reaction of glycine with O-ftaldialdheyde and mercaptoethanol to produce a fluorescent derivative 1- (2'-hydroxyethylthio) -2-N-alkyl-lisoindole which is measured by a fluorescence detector with excitation of 340 nm and emission of 455 nm wavelenghts. A factorial design 2^3 was applied to establish the best conditions to detect Glyphosate and AMPA with the variables: HClO- concentration (X1), coil temperature (X2) and flow used in the OPA-MERC pump (X3). The influence of selected factors and their two and three interactions in the detection of Glyphosate and AMPA was studied. The best relationship between the studied variables was flow of 0.5 mL min-1, 38 °C and concentration of 20 mg L-1.