A method for the determination of hormones in honey was developed using an Agilent 6460 LC/MS/MS with an Agilent Poroshell 120 EC-C18 column. At the same time a new sample preparation method using Agilent Bond Elut Plexa was developed and optimized for extracting eight Macrolides including Leucomycin, Oleandomycin, Erythromycin, Tylosin, Tilmicosin, Roxithromycin, Spiramycin and Josamycin in honey. Results indicate that the method well separated the eight macrolides. The Agilent Poroshell 120 EC-C18 column (3.0 mm ×100 mm, 2.7 μm) provided a quick separation with symmetrical peak shapes and high performance. The Agilent Bond Elut Plexa for sample preparation can be used for purification and enrichment of Macrolides in complex matrix like honey. The recovery and reproducibility based on matrix spiked standards are acceptable for macrolides residues in honey.