'D**k in a Box', a spoof of R&B love songs, premiered on a 2006 edition of SNL and later went on to garner millions of views on the internet.
A sequel called 'Motherlover' - featuring Susan Sarandon and Patricia Clarkson as love interests - aired during Timberlake's third hosting stint in 2009.
TMZ has now obtained photos of Samberg and Timberlake filming a third instalment to air during Saturday Night Live's season finale.
Clarkson and Sarandon were also spotted shooting scenes in New York City for the 'D**k in the Box' reprise, once again portraying Samberg and Timberlake's lovers.
Timberlake has been a frequent guest on the sketch comedy series in recent years, having made more than a half-dozen cameo appearances aside from his hosting turns.
SNL head-writer and 'Weekend Update' anchor Seth Meyers recently professed that he's excited to see Timberlake and musical guest Lady GaGa return to the NBC late-night series to wrap the 36th season.
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Watch the 'D**k in a Box' and 'Motherlover' sketches below: