Therapeutic drug monitoring for immunosuppressants is a widely conducted global practice. Traditionally, the pretreatment of whole blood involves the use of metal ions combined with organic solvents. However, this method requires multiple reagent additions, repeated opening, closing, and vortexing of vials, and it also leads to heavy metal pollution. Given the typically large sample volumes, optimizing this process is crucial for increasing throughput, reducing the workload of clinical staff, and lowering costs. We discovered that treating whole blood with a 60 to 75% acetonitrile (ACN) solution effectively releases tacrolimus, sirolimus, and cyclosporine A while simultaneously precipitating protein. This allowed us to significantly simplify the pretreatment process to just adding 65% ACN solution containing internal standards, manually shaking for 20 s, and centrifuging for 2 min. The resulted supernatant can then be directly analyzed by mass spectrometry. Method validation demonstrated that the new approach can accurately quantify tacrolimus in the range of 0.64 to 37.5 ng/ml, cyclosporine A at 12 to 976 ng/ml, and sirolimus at 0.99 to 43.4 ng/ml. A comparison of paired samples showed the new method to be perfectly consistent with the classical method, with 293 out of 300 results deviating by no more than ± 20%. This study has greatly simplified the workflow, increased throughput, and resolved environmental concerns for therapeutic drug monitoring of immunosuppressants, including tacrolimus, sirolimus, and cyclosporine A, in whole blood samples. The proposed method is a viable replacement for existing protocols and deserves to be adopted in all clinical laboratories with relevant practical needs globally.