The main objective of the cleaning validation procedure is to verify the effectiveness of the cleaning procedure for removal and minimising the risk of cross-contamination, a topic that has become more important regarding the development of the medicines. Furthermore, if a product is found to be the worst among many of the products, one cleaning validation procedure of the worst-case product can cover the validation of the remaining ones, thus saving time and money. A novel, reproducible and efficient high-performance liquid chromatography (HPLC) method was optimised and validated for the detection of the following cephalosporin residues: cephalexin (CPH), cefaclor (CFC), cefixime (CFX), cefdinir (CFR) and ceftazidime (CFZ) in human spiked plasma and in production machines using rinse and swab sampling collected from surfaces and application to Cosa®CIP Detergent. Isocratic chromatographic system was performed at ambient temperature using mobile phase consisting of acetonitrile: 40% tetrabutylammonium hydroxide adjusted to pH 7.0 ± 0.1 with 10% phosphoric acid (72.5:27.5, v/v) on Ultrasphere ion pair column (250 mm × 4.6 mm, 5.0 μm particle size) at a flow rate 1.0 mL/min, injection volume 10 μL and UV detection at 265 nm. The chromatographic run time was less than 20 min for the mixture. Linear relationships were obtained over the concentration ranges 0.5–25 ng mL−1 for CPH, 1.5–30 ng mL−1 for CFC, 2–33 ng mL−1 for CFX, 3–35 ng mL−1 for CFR and 4–40 ng mL−1 for CFZ with correlation coefficients >0.998. Analytical and bioanalytical validation methods were carried out following terms of linearity, specificity, LOQ, LOD, accuracy and precision for determination of cephalosporin residues in production machines and in human spiked plasma according to FDA guidelines. [ABSTRACT FROM AUTHOR]