Abiraterone acetate is an approved prodrug administered orally in a fixed dose format for the treatment of metastatic castration-resistant prostate cancer (mCRPC). In vivo, the prodrug is readily metabolized to abiraterone and its active metabolite Δ(4)-abiraterone (D4A) which selectively and irreversibly inhibit the 17α-hydroxylase/17,20-lyase (CYP17A1) enzyme and the androgen receptor, respectively. Therapeutic drug monitoring (TDM) of abiraterone and its metabolites may be beneficial as significant pharmacokinetic variability has been observed. Dried plasma spots (DPS) represent an attractive, yet under-utilised approach for TDM analysis with desired features including easy collection, transport, storage and overcomes the issues of blood hematocrit levels known in dried blood spot analysis. In this study we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous quantification of abiraterone and D4A with deuterated internal standard (abiraterone D4) from DPS using a high-resolution benchtop mass spectrometer. Calibration curves were linear over a wide and clinically-relevant concentration range (0.132-196.0 ng/mL for abiraterone and 0.110-39.17 ng/mL for D4A) with high accuracy (93-104% for abiraterone and 96-108% for D4A) and precision (%CV ≤ 12.5). As expected, the levels of abiraterone and D4A obtained from DPS from mCRPC patients varied substantially (1.5-31.4 ng/mL for abiraterone and 0.1-5.2 ng/mL for D4A; n = 22). Detailed benchmarking of the DPS method to a pre-validated liquid plasma method showed that the techniques generate quantitative indistinguishable data. Collectively, this demonstrates the potential of using LC-MS/MS in combination with DPS for TDM of abiraterone and D4A from patients.