Due to narrow therapeutic indices of immunosuppressants and highly variable pharmacokinetics, therapeutic drug monitoring is necessary to individualize immunosuppressant dosage following organ transplantation. Until recently, monitoring was performed primarily using immunoassays; however, there is an increasing shift to HPLC coupled with MS/MS, due to its greater sensitivity and specificity. Online sample clean-up with either a single analytical column or with 2D chromatography significantly reduces manual handling and is essential to minimize matrix effects and maximize specificity. Coupled with rapid chromatography, it allows simultaneous analysis of the major immunosuppressants, with high sample throughput. Thus, LC-MS/MS is an attractive and versatile technique that facilitates rapid development of analytical methods, including new immunosuppressants as they become approved for clinical use.