• Tetracyclines were extracted from food matrices and human plasma by EME. • Optimal extraction parameters differed depending on the sample matrix. • Good analytical performance was achieved for all analytes across all matrices. • EME provided an efficient, selective, and green alternative to traditional methods. • The greenness of the method was evaluated by the AGREEprep metric tool. Tetracyclines are widely used antibiotics in animal farming, raising concerns over their residues in food and associated health risks. Current methods for residue determination are complex, time-consuming, and produce substantial solvent waste. Electromembrane extraction (EME) offers high clean-up efficiency, minimal solvent use, and direct LC-MS/MS compatibility. This study evaluates EME for extraction of four tetracyclines (oxytetracycline, tetracycline, chlorotetracycline, doxycycline) from complex matrices including milk, egg white, honey, and human plasma. Optimal parameters varied across matrices, underscoring the need for matrix-specific optimization. Optimized methods achieved recoveries of 66–101 %, excellent linearity (R² = 0.9929–0.9998), and minimal matrix effects (-14 % to 1 %). LLOQs ranged from 0.5 to 2.1 ng/g, with calibration curves extending to 1000 ng/g, covering concentrations relevant for residue monitoring. These results demonstrate EME’s potential for reliable analysis of challenging polar and zwitterionic compounds in food and biological samples. Finally, the greenness was evaluated by the AGREEprep metric tool, gaining a total score of 0.64.