Elsewhere Maccabi Haifa joined the lucrative stages of Europe´s premier club competition with a 3-0 win over FC Salzburg which gave the Israelis a 5-1 victory over the Austrians.
Debrecen secured their ticket in similar fashion, becoming the first Hungarian side to qualify for the group stages since Ferencvaros in 1995 by beating Bulgarian outfit Levski Sofia 2-0 at home on the night for an aggregate of 4-1.
Ventspils had been hoping to become the first Latvian side to qualify for the group stages however despite scoring a goal at FC Zurich they were defeated 2-1, for an aggregate score of 5-1.
Atletico Madrid had taken a major stride towards qualification with a 3-2 win away at Panathinaikos last week and confirmed their superiority over the Greeks with a 2-0 win in Madrid which puts them among the last 32 for the second year running.
In the past 10 years Lyon have been regulars in the group stages, reaching the quarter-finals in 2006 when they were eliminated by AC Milan.
Having lost the French league title for the first time in eight seasons in May, to Bordeaux, the French giants were forced to come through qualifying.
But despite predictions of a close two-leg fixture against Anderlecht, Claude Puel´s side showed they more than deserve to join Europe´s elite.
After their 5-1 mauling of the Belgians last week in Lyon, where Lisandro scored their second goal from a penalty in the 15th minute, Anderlecht had a mountain to climb in this second leg.
Lining up alongside fellow Argentine Cesar Delgado, on the left wing, Lisandro bounced back in style from a groin strain at the weekend to put the French side into the lead with a superb lob in the 25th minute following an error in the hosts´ defence.
Barely seven minutes later the former FC Porto striker pounced on Anthony Reveillere´s pass to double his score.
Lisandro added a third four minutes before half-time after pouncing on a rebound from Delgado, the Argentine later being substituted on the hour mark for 17-year-old Ishak Belfodil.
The loss sees Anderlecht drop down to the Europa League.
Lyon president Jean-Michel Aulas said: "That was an achievement. We thought we could qualify but at the same time very difficult up against a pretty strong anderlecht side and they´ll prove that in the Europa League."
"As we saw last week Lyon were very strong," said Anderlecht coach Ariel Jacobs.
"In the first leg we laid out the red carept for them, today we offered them nice presents: two goals out of three! If we can get away with certain mistakes in the Belgian league we can´t do that in the Champions League."
Atletico manager Abel Resino meanwhile reflected on a job well done against Panathinaikos.
"Our objective wasn´t to put on a great match but to make it into the group stage. Occasionally we lacked fluidity in our play but overall we had a serious game, which was the more important."
Wednesday´s second leg play-offs feature Arsenal aiming to drive home their 2-0 first leg advantage over Celtic at the Emirates Stadium.
Second Leg Results of the Champions League play-offs on Tuesday
At Budapest: Debrecen (HUN) 2 (Varga 13, Rudolf 35) Levski Sofia (BUL) 0 (Debrecen qualify 4-1 on aggregate)
At Brussels: Anderlecht (BEL) 1 (Suarez 51-pen) Lyon (FRA) 3 (Lisandro 26, 32, 41) (Lyon qualify 8-2 on aggregate)
At Haifa, Israel: Maccabi Haifa (ISR) 3 (Dvalishvili 31, Golasa 57, Ghadir 90) FC Salzburg (AUT) 0 (Maccabi qualify 5-1 on aggregate)
At Madrid: Atletico Madrid (ESP) 2 (Vintra 4-og, Aguero 83) Panathinaikos (GRE) 0 (Atletico qualify 5-2 on aggregate)
At Zurich: FC Zurich (SUI) 2 (Vonlanthen 6, Abdi 90+2) FK Ventspils (LAT) 1 (Tigirlas 8) (Zurich qualify 5-1 on aggregate)
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