The objective of this study was to develop a validated, transient, chemically induced lameness model in sows using subjective and objective lameness detection tools. Experiment 1 determined an effective joint injection technique based on volume and placement of dye using feet collected from 9 finisher pigs and 10 multiparity cull sow carcasses. Experiment 2 confirmed the injection technique in live animals and produced a transient clinical lameness in 4 anesthetized sows injected with amphotericin B (15 mg/mL) in the distal interphalangeal joints of the claw. Clinical lameness was assessed by a categorical lameness scoring system, and a postmortem visual confirmation of joint injection technique was obtained. In Exp. 3, 6 sows were injected with 0, 10, or 15 mg/mL amphotericin B in either the left or right hind foot and were monitored until clinical resolution. Treated sows demonstrated elevated clinical lameness scores. These changes resolved by 7 d after lameness induction. Control sows injected with sterile saline developed a clinical lameness score of 0.5, which resolved 72 h post injection. In Exp. 4, 36 sows were injected with 10 mg/mL amphotericin B in 1 of 4 injection sites (left front claws, right front claws, left rear claws, and right rear claws). All injected sows exhibited a decrease in maximum pressure, stance time, and number of sensors activated on the GaitFour (P < 0.05) sensor system. A static force plate also demonstrated a decrease in weight (kg) being placed on the injected foot when all feet were injected (P ≤ 0.05). Injection of amphotericin B induced a predictable acute lameness that resolved spontaneously and is an effective method to model lameness in sows.