However, one of the important limitations of method based on spectrophotometer, chemiluminescence or spectrofluorimetry is the fact that these compounds are inactive species for direct detection. Therefore, the derivatization procedure is normally required that make the methods are tedious, expensive, and long time analysis. The high sensitivity and selectivity of electrochemical detection are desired for antibiotics determination. Electrochemical techniques are alternatives, which can be cheap, fast and simple. The working electrode, mercury, is extensively used for determination tetracyclines [23,24]. This electrode has some problems such as the toxicity and limited stability of responses. In recent years, the flow injection system has received much attention and some analytical applications have been reported. A flow injection system was introduced to conventional analytical instrument to improve sample throughput and sensitivity that are the requirement to develop the assay method in pharmaceutical industry. Thus, use of the flow injection system coupled to the mercury electrode is complicated. Moreover, problems associated with easily oxidized mercury electrode have to be considered. Voltammetry and amperometry are the techniques that offer the high sensitivity. Their disadvantage is deposition of the detection product or solution impurities on the electrode surface. Therefore, pulsed amperometric detection with alternated anodic and cathodic polarization to clean and reactivate the electrode surface, has been introduced to overcome this problem. PAD offers the possibility to clean and reactivate the electrode surface effectively after measuring cycle without mechanical polishing [25–28]. In the simplest implementation of PAD, the potential of the working electrode is stepped between the potentials for detection, Edet, cleaning, Eoxd and reactivation, Ered. All three steps of PAD require the following: (a) the oxidation of analyte during the detection step; (b) the oxidative desorption of adsorbed detection products or solution impurities at the cleaning step; (c) the cathodically dissolved of inert oxide product during reactivation step [29]. Pulsed amperometric detection has been used for the sensitive detection of numerous compounds [30–34]. It also has been successful for the determination of tetracycline in pharmaceutical formulation [35]. The goal of this work is extended the use of the PAD waveform for the determination of doxycycline or chlortetracycline in pharmaceutical formulations. In this research also employed the flow injection system with PAD to reduce time analysis and to obtain low detection limit. The present method has been proved to be simple, rapid, sensitive and suitable for automatic analysis.