IQG-607 is an analog of isoniazid with anti-tuberculosis activity. This work describes the development and validation of an HPLC method to quantify pentacyano(isoniazid)ferrate(II) compound (IQG-607) and the pharmacokinetic studies of this compound in mice. The method showed linearity in the 0.5–50 μg/mL concentration range (r = 0.9992). Intra- and inter-day precision was <5%, and the recovery ranged from 92.07 to 107.68%. IQG-607 was stable in plasma for at least 30 days at −80 °C and, after plasma processing, for 4 h in the auto-sampler maintained on ice (recovery >85%). The applicability of the method for pharmacokinetic studies was determined after intravenous (i.v.) and oral (fasted and fed conditions) administration to mice. IQG-607 levels in plasma were quantified at time points for up to 2.5 h. A short half-life (t1/2) (1.14 h), a high clearance (CL) (3.89 L/h/kg), a moderate volume of distribution at steady state (Vdss) of 1.22 L/kg, were observed after i.v. (50 mg/kg) administration. Similar results were obtained for oral administration (250 mg/kg) under fasted and fed conditions. The oral bioavailability (F), approximately 4%, was not altered by feeding. Plasma protein binding was 88.87 ± 0.9%. The results described here provide novel insights into a pivotal criterion to warrant further efforts to be pursued towards attempts to translate this chemical compound into a chemotherapeutic agent to treat TB.