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Background: Hematocrit is usually measured from venous blood collected by invasive venipuncture. This study was performed to determine hematocrit accurately and precisely using minimally invasive volumetric absorptive microsampling (VAMS) technique. Such technique is to be applied to determining hematocrit in various clinical settings for the care, including therapeutic drug monitoring, of neonatal or epileptic patients, or patients with high risk of infection or bleeding. Methods: The study was performed using 31 VAMS samples obtained from 21 pancreatic cancer patients. Hematocrit was determined using the values of potassium concentrations obtained from blood in VAMS tips (HctVAMS). HctVAMS was compared with hematocrit measured from blood collected by venipuncture (HctVP). The accuracy and precision of HctVAMS in comparison to HctVP were evaluated using Bland- Altman plot, Deming regression and mountain plot. Results: Bland-Altman plot displayed a random scattering pattern of the differences between HctVAMS and HctVP with the mean bias of −0.010 and the 95% limit of agreement ranging from −0.063 to 0.044. Deming regression for HctVAMS and HctVP line demonstrated very small proportional and constant biases of 1.04 and −0.003, respectively. Mountain plot exhibited a narrow and symmetrical distribution of the differences with their median of −0.011 and central 95% range from −0.049 to 0.033. Conclusion: Hematocrit was accurately and precisely determined using less invasive VAMS technique. Such technique appears to be applicable to determining hematocrit in situations that venipuncture is not favorable or possible.