Third‐generation cephalosporins are semi‐synthetic antibiotics with enhanced activity against Gram‐negative organisms. The cephalosporins in biological samples are mostly determined using high‐performance liquid chromatography (HPLC) and HPLC–mass spectrometry (LC‐MS) or tandem mass spectrometry (LC‐MS/MS). In recent years, numerous bioanalytical methods have been developed to improve the sensitivity and specificity of cephalosporin quantification using the powerful LC‐MS/MS systems that are common in research laboratories. This review article presents recently developed bioanalytical methods by HPLC or LC‐MS(/MS) for 12 third‐generation cephalosporins, including five oral third‐generation cephalosporins, and further discusses their application in pharmacokinetic studies of animals and humans. Copyright © 2014 John Wiley & Sons, Ltd.