Background: Lamivudine is widely used alone or in combination with other anti-HIV drugs in the infant to adolescent age groups of pediatric populations. Compounding of medications is frequently used for pediatric patients. However, many issues have been reported for the compounded formulation such as assay, stability, safety, and efficacy. Three-dimensional printing can overcome these issues. Objective: The aim of this study was to understand the effect of process and formulation variables on lamivudine printlets for pediatric populations using selective laser sintering. Methods: The Plackett–Burman screening design was used to prepare 12 formulations to study six variables, namely, laser scanning speed (130–150 °C), surface temperature (105–120 °C), chamber temperature (250–350 mm/s), sucrose (0–30%), hydroxypropyl methylcellulose (0–42%), and Kollidon^® CL-M (0–5%). The formulations were tested for dissolution, disintegration, hardness, assay, X-ray diffraction, differential scanning calorimetry, stability, and pharmacokinetics in Sprague Dawley rats. Results: The assay of the printlet formulations varied between 93.1 and 103.5% and the disintegration time was 2.8 ± 1.2 (F1) to 43.7 ± 2.7 (F10) s. Due to high surface temperatures, the unsintered powder in the printing chamber experienced significant changes in crystallinity. No statistical significance was observed between the pharmacokinetic parameters of the printlets and commercial tablets (p > 0.05). The maximum plasma concentration (C_max), time to reach maximum plasma concentration (T_max), and area under the curve (AUC) of the printlets and commercial tablets were 295.5 ± 33.0 and 305.0 ± 70.1 ng/mL, 0.5 ± 0.0 and 1.0 ± 0.8 h, and 1414.1 ± 174.0 and 1987.2 ± 700.5 ng.h/mL, respectively. Conclusions: In summary, fast-disintegrating and dissolving 3D printed lamivudine was found to be bioequivalent to commercial formulation of lamivudine. Thus, it is a viable method for dispensing personalized lamivudine printlets for pediatric populations.