Chimeric urokinase type plasminogen activator (uPA)/severely severe combined immunodeficiency (SCID) mice reconstituted with humanized livers are useful for studying hepatitis B virus (HBV) infection in the absence of an adaptive immune response. However, the detailed characterization of HBV infection kinetics necessary to enable in-depth mechanistic studies in this in vivo HBV infection model is lacking. To characterize HBV kinetics post-inoculation (p.i.) to steady state, 42 mice were inoculated with HBV. Serum HBV DNA was frequently measured from 1 minute to 63 days p.i. Total intrahepatic HBV DNA, HBV covalently closed circular DNA (cccDNA), and HBV RNA was measured in a subset of mice at 2, 4, 6, 10, and 13 weeks p.i. HBV half-life (t1/2 ) was estimated using a linear mixed-effects model. During the first 6 hours p.i., serum HBV declined in repopulated uPA/SCID mice with a t1/2 = 62 minutes (95% confidence interval [CI] = 59-67). Thereafter, viral decline slowed followed by a 2-day lower plateau. Subsequent viral amplification was multiphasic with an initial mean doubling time of t2 = 8 ± 3 hours followed by an interim plateau before prolonged amplification (t2 = 2 ± 0.5 days) to a final HBV steady state of 9.3 ± 0.3 log copies (cps)/mL. Serum HBV and intrahepatic HBV DNA were positively correlated (R2 = 0.98).