Cephalothin is a first-generation cephalosporin, that shows great activity against Gram-positive microorganisms. Its effect is bactericidal, and due its action, it is the most efficient first-generation cephalosporin against resistant microorganism (β-lactamase producers). Although this drug has been clearly studied and researched about its antimicrobial activity, pharmacokinetics and pharmacodynamics, there are a few studies in literature regarding the development of analytical methodology for this cephalosporin. The aim of this work was to develop and validate a new method of analysis, using high performance liquid chromatography, resulting in an innovative method, quick and using solvents of low toxicity, minimizing, in this way, its toxic actions to the operators and leavings in the environment. The method developed and validated for the quantification of sodium cephalothin in lyophilized powder for injectable solution used high performance liquid chromatography (HPLC). The mobile phase consisted in water with 0.7% of glacial acetic acid and ethanol (70:30 v/v), wave-length of 237 nm, Zorbax Eclipse Plus C18 AgilentTM column and room temperature of 25°C, retention time of 4,20 minutes. The method was linear in the concentrations of 20, 40, 60, 80 e 100 μg/mL, selective, accurate and robust towards these modifications: ethanol brand, water source, mobile phase rate, glacial acetic acid proportion, flow rate, room temperature and wave length. The dosing for CET was of 106.72%.