Cefazolin is a first-generation cephalosporin used to treat severe infections of the respiratory tract, urinary tract, skin, and soft tissues. This study presents the optimal conditions for the determination of cefazolin by thin-layer chromatography with densitometric detection. A chloroform−methanol−glacial acetic acid mixture (6:4:0.5, v/v/v) was selected as the mobile phase, while TLC silica gel 60F254 plates were used as the stationary phase. Next, the developed procedure was validated in accordance with ICH guidelines. The obtained results showed that the method is selective, precise, and accurate in a linearity range of 0.04−1.00 µg/spot (r > 0.99). Subsequently, qualitative and quantitative analyses of formulations containing cefazolin were performed. It was found that the amount of antibiotic is highly consistent with the content declared by manufacturers. The suitability of the developed method for stability testing under varying environmental conditions was also verified. It was found that under the tested conditions, the degradation process follows first-order kinetics. The lowest stability was registered in an alkaline environment and in the presence of an oxidizing agent, and the highest stability was recorded in water, and these results were confirmed by the calculated kinetic parameters. The developed method can be used in qualitative and quantitative analyses and stability studies of the analyzed antibiotic.