A simple and effective method was developed for the simultaneous determination of chlortetracycline (CTC), oxytetracycline (OTC), tetracycline (TC) and doxycycline (DC) in beehives using liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS). Analytes were extracted in EDTA-McIlvaine buffer from beehives by ultrasonication and shaking, purified using hexane distribution and HLB cartridges, quantified using a matrix-matched standard calibration curve, and validated according to Commission Decision 2002/657/EC and SANCO/10684/2009. The responses were linear in the range of 1 to 500 mg L-1 amount to concentration of 0.2 to 100 mg kg-1 based on 5 g of sample with correlation coefficients (r2)>0.99. The overall recoveries were in the range of 73.8 to 106.7% with RSD of 1.19 to 9.8%. The limits of quantification (LOQ) were 1 mg kg-1 for CTC and DC and 0.2 mg kg-1 for OTC and TC. The decision limits (CCa) ranged from 0.041 to 0.31 mg kg-1, and the detection capabilities (CCb) ranged from 0.064 to 0.43 mg kg-1. The application of this method revealed low concentrations of OTC and TC in some real beehive samples. The results showed that the developed method is sensitive and accurate and can be used to determine the levels of tetracycline antibiotics in beehives.