The objective of this study was to evaluate the effect of collagenase and two final dimethylformamide (DMF) concentrations (4% and 7%) on alpaca frozen-thawed sperm quality. A total of 25 ejaculates from 5 alpaca were obtained using electroejaculation. Each individual ejaculate was evaluated and then diluted 4:1 in a solution of 1 mg/ml collagenase in HEPES-TALP medium and incubated for 4 min at 37°C. Subsequently, samples were diluted in TRIS-fructose-citric acid-egg yolk and cooled to 5°C. Then, each sample was divided in two aliquots and DMF at final concentration of 4% or 7% was added, equilibrated for 1 hr at 5°C and frozen over liquid nitrogen vapours. A Kruskal–Wallis test was used to evaluate the sperm morphometry, and Completely Random Block designs were used to analyse sperm motility, viability, membrane function and acrosome status. After collagenase incubation, none of the samples showed thread formation, and sperm parameters were preserved. Non-progressive motile sperm were higher (p <.05) in equilibrated samples (4% DMF: 31.8 ± 8.3% and 7% DMF: 36.3 ± 11.8%) compared to raw (10.1 ± 4.3%) and frozen-thawed semen (4% DMF: 9.7 ± 1.8% and 7% DMF: 7.5 ± 3.2%). Sperm membrane function, membrane integrity and intact acrosomes were higher (p <.05) in raw semen (40.1 ± 12.2%, 94.6 ± 3.2% and 91.3 ± 8.1%) compared to frozen-thawed samples (4% DMF: 19.8 ± 4.7%, 53.2 ± 2.7%, 65.7 ± 8.7% and 7% DMF: 20.4 ± 4.5%, 54.1 ± 1.4%, 64.6 ± 9.1%). Length of the sperm head was lower in frozen-thawed samples, being statistically different with 4% DMF compared to pre-freezing samples. The ratio between acrosome and head areas was greater (p <.05) in frozen-thawed samples. Incubation of raw alpaca semen with collagenase decreased the thread formation without affecting sperm quality. Frozen of collagenase treated alpaca semen with 4% or 7% DMF did not preserve the sperm parameters in thawed samples.