For decades, chloroquine has been the drug of choice for the treatment of malaria. It is safe, cheap and widely available, making it ideal for mass drug administration. Its extensive use resulted in rapid and widespread development of resistant parasites and it has been rendered useless for the treatment of P. falciparum malaria. However, it remains highly effective against P. vivax malaria in most areas and it is currently the frontline drug for the treatment of blood stage P. vivax infections. Therefore, it is crucial to develop sensitive and accurate tools to measure the drug in biological fluids in order to study its pharmacokinetic and pharmacodynamic properties. Here we present a novel set of methods to quantify chloroquine and its metabolite, desethylchloroquine, in various patient samples. Different high-throughput extraction techniques were used to recover the drug from plasma (100 µL), whole blood (50 µL), and whole blood (50 µL) applied on filter paper followed by quantification with LC-MS/MS. The developed method showed excellent separation of compounds a very short analysis time of 6 min per clinical sample. The highly sensitive approach allowed quantification of chloroquine and desethylchloroquine in patient samples in concentrations as low as 1.41 ng/mL. In conclusion, the presented methods were fast, sensitive, and accurate. The small volumes of biological fluids required, allows the implementation in clinical pharmacokinetic studies in children and other vulnerable patients. Whole blood applied on filter paper (dried blood spot) is a field adapted sampling strategy, enabling pharmacokinetic studies in rural areas where centrifugation and cold-chain transport are difficult.