Cardiotonic steroids (CTSs), such as digoxin, are used for heart failure treatment. However, digoxin permeates the brain-blood barrier (BBB), affecting central nervous system (CNS) functions. Finding a CTS that does not pass through the BBB would increase CTSs' applicability in the clinic and decrease the risk of side effects on the CNS. This study aimed to investigate the tissue distribution of the CTS ouabain following intraperitoneal injection and whether ouabain passes through the BBB. After intraperitoneal injection (1.25 mg/kg), ouabain concentrations were measured at 5 min, 15 min, 30 min, 1 h, 3 h, 6 h, and 24 h using HPLC-MS in brain, heart, liver, and kidney tissues and blood plasma in C57/black mice. Ouabain was undetectable in the brain tissue. Plasma: C max = 882.88 ± 21.82 ng/g; T max = 0.08 ± 0.01 h; T 1/2 = 0.15 ± 0.02 h; MRT = 0.26 ± 0.01. Cardiac tissue: C max = 145.24 ± 44.03 ng/g (undetectable at 60 min); T max = 0.08 ± 0.02 h; T 1/2 = 0.23 ± 0.09 h; MRT = 0.38 ± 0.14 h. Kidney tissue: C max = 1072.3 ± 260.8 ng/g; T max = 0.35 ± 0.19 h; T 1/2 = 1.32 ± 0.76 h; MRT = 1.41 ± 0.71 h. Liver tissue: C max = 2558.0 ± 382.4 ng/g; T max = 0.35 ± 0.13 h; T 1/2 = 1.24 ± 0.7 h; MRT = 0.98 ± 0.33 h. Unlike digoxin, ouabain does not cross the BBB and is eliminated quicker from all the analyzed tissues, giving it a potential advantage over digoxin in systemic administration. However, the inability of ouabain to pass though the BBB necessitates intracerebral administration when used to investigate its effects on the CNS.