Commercially available radioimmunoassays (RIA) with ¹²⁵I-labeled hormones were adapted, developed and validated to quantify progesterone and testosterone in gonad extracts of Octopus maya, considered an easily domesticated species with potential in aquaculture. Development of the RIAs was divided into four phases: (1) extraction of progesterone and testosterone from gonads, (2) preparation of gonad extracts for RIA, (3) standards preparation, and (4) RIA procedure. Hormones were extracted twice with 15 mL of diethyl-ether each time. The recovered hormone and the solvent effect on extraction were evaluated. RIAs were validated based on specificity, sensitivity, accuracy, precision and reproducibility. Serially diluted gonad extracts were parallel to the standard curves of each hormone, indicating specificity. Sensitivities for both hormones were less than 0.1 ng·g^-1, and did not change by more than 20% during the ¹²⁵I half-life. Accuracy was 102.25 ± 11.22% for progesterone and 81.62 ± 6.24% for testosterone. For both hormones, precision was close to 13% and reproducibility 17%. The precision/reproducibility ratio indicated a system with good stability. The amounts of the hormones recovered increased when the number of extractions was increased. Hormone concentrations also increased when the volume of diethyl-ether used per extraction was increased. The range of progesterone and testosterone concentrations quantified in this study is in agreement with the range found in other octopuses. Finally, the developed and validated RIAs meet the requirements for quantification of progesterone and testosterone in Octopus gonad extracts and show that it is possible to use ¹²⁵I-labeled hormone as well as the plastic coated-tubes from commercial RIA kits to analyse variations in hormone levels associated with gonad developmental stages and the reproductive cycle of octopuses, allowing exploration of their possible functions.