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Our work was to develop a methodology to qualitatively and quantitatively detect tetracyclines (TCs) derivatives, such as doxycycline (DOTC) and oxytetracycline (OTC). The Raman spectra region of 500-1800 cm-1 reveals distinctive spectra features of TCs. We applied principal component analysis (PCA) to investigate the antibiotics composition from Raman spectra. The capability of the model was evaluated by partial least square analysis (PLS) with a leave-one-out-cross-validation method. The result shows the feasibility of simultaneous quantitative detection of tetracycline derivatives by portable Raman spectroscopy.